Method for detecting chromosome Robertsonian translocation

ABSTRACT

Provided is a method for detecting chromosomal Robertsonian translocation and the SNP (single nucleotide polymorphism) site and primer composition for use therein. The method is practical, simple, convenient and high in universality; moreover, a high-throughput sequencing technology is combined, so that the method has the advantages of low cost, and high sensitivity, specificity and accuracy.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a national phase entry under 35 U.S.C. § 371 of International Application No. PCT/CN2016/070696, filed on Jan. 19, 2016, the content of which is hereby incorporated by reference in its entity.

SEQUENCE LISTING

The present application contains a Sequence Listing which has been submitted via EFSWeb and is hereby incorporated by reference in its entirety. The ASCII copy, created on Jul. 9, 2018, is named 00242_0001sequencelisting.txt, and is 156 kilobytes in size.

TECHNICAL FIELD

The present disclosure relates to molecular diagnosis and particularly relates to a method for detecting chromosomal Robertsonian translocation, which is capable of detecting normal and translocation carrying embryo and fetus, of which the parents have pedigree of Robertsonian translocation.

BACKGROUND

Robertsonian translocation (ROB) is named according to the American biologist William Rees Brebner Robertson who for the first time found the translocation pattern and is a chromosomal translocation mainly occurring in 5 acrocentric chromosomes (Chromosome 13, Chromosome 14, Chromosome 15, Chromosome 21 and Chromosome 22) (Robertson WRB. Chromosome studies. I. Taxonomic relationships shown in the chromosomes of Tettigidae and Acrididae. V-shaped chromosomes and their significance in Acrididae, Locustidae and Gryllidae: chromosome and variation. J Morph 1916, 27: 179-331). When two acrocentric chromosome break at centromere or a location in proximity to centromere, their long arms bind at centromere forming a derived chromosome composed of long arms, which is also known as centromere fusion, one of chromosomal balanced translocations. Two short arms form a small chromosome, which always loses in a second division. Robertsonian translocation is a common chromosomal abnormal structure, of which the newborn occurrence rate is 1.23/1000 (Nielsen J, Wohlert M. Chromosome abnormalities found among 34910 newborn children: results from 13-year incidence study in Arhus, Denmark. Hum Genet. 1991, 87: 81-83). As the short arm of chromosome mainly comprises random repeated DNA and ribosome RNA genes, which are present in all of the five chromosome, there is loss of short arm of chromosome in Robertsonian translocation, it does not results in pathogenic phenotype. Although the phenotype is normal, during the Pt meiosis of carrier's germ cell, the translocation chromosome and two corresponding normal chromosome will match to form trivalent chromosome and such a structure will result in three division patterns, i.e. alternate, neighborhood and uncommon 3:0 division patterns, of which only the alternate pattern will lead to normal or balanced gametes and others will lead to unbalanced gametes. The dominating unbalanced gametes will lead to difficulty in pregnancy or recurrent abortion in pregnancy, and even lead to birth of children with congenital defect like 13-Patau syndrome with abnormal chromosome copy number in translocation carrier. Now more and more Robertsonian translocation carriers seek for test-tube baby technology for preimplantation genetic diagnosis.

The test-tube baby is a technology that ovum and sperm are taken to put in a special culture liquid for culture, fertilization, and the oosperm develops into an embryo in a thermostat incubator, which is then transplanted into parent uterus where it develops into a fetus finally.

A key of successful prevention lies in selecting health embryo for transplant. Preimplantation genetic screen refers to detection of chromosome number and abnormal structure before implantation and nidation of an embryo, selection for an embryo with normal chromosome for implantation into uterus, so as to give normal progeny. The current preimplantation genetic diagnosis for Robertsonian translocation comprises SNP chip and fluorescence in situ hybridization (FISH). The SNP chip is the most commonly used detection technology, which however can only detect embryo chromosome copy number change but cannot differentiate normal embryo from translocation carrying translocation in Robertsonian translocation. FISH uses specific nucleic acid probe labeled with fluorescence to hybrid with corresponding target DNA molecule in cells, and fluorescence signal is observed under fluorescence microscope to determine the location of DNA molecule bound with fluorescence probe in chromosome. Nevertheless, FISH as a method for examine single nucleus has some defect as 7% false negative rate (Colls P, Escudero T, Cekleniak N, Sadowy S, Cohen J, Munné S. Increased efficiency of preimplantation genetic diagnosis for infertility using “no result rescue”. Fertil Steril. 2007, 88(1): 53-61).

Spontaneous abortion is a common disease in gynaecology and obstetrics, of which the occurrence rate account for 10%-15% of all the pregnancy. The occurrence rate is determined by many factors, like embryo factor, placenta factor, parent factor, environmental factor or the like, wherein abnormal embryo chromosome is the main cause of abortion.

Chromosomal examination on abortion tissue for the causes of embryo developmental arrest, abortion provides theoretical basis for clinical counsel and guideline for subsequent pregnancy. Now FISH technology is for use in performing common abnormal chromosome on missed abortion villus and tissues at different locations of a fetus. FISH as a targeted detecting technology has limited commercial probes and was limited by the type, amount and high cost of the probes. It cannot detect a large number of abnormal genes or chromosome. Additionally, due to the limit of FISH, it cannot be used to detect chromosomal abnormal structure, leading to missed diagnosis of some patients with chromosomal abnormal structure (L I, Yangyang, Zhang Qin, Current study of analysis of spontaneous abortion villus chromosome karyotype, Journal of Obstetrics and Gynaecology, 2012, 28: 431-433).

In a summary, it is desired to develop a novel method to detect Robertsonian translocation of embryo and abortion tissue in test-tube baby technology.

SUMMARY

Provided is a method for detecting chromosomal Robertsonian translocation based on high-throughput sequencing technology so as to screen fully normal and translocation carrying embryo, fetus or abortion tissue.

In the first aspect, provided is a method for detecting chromosomal Robertsonian translocation, comprising the steps of:

(1) obtaining DNA samples from both of the parents and the progeny;

(2) screening hypermutational SNP site from a location in proximity to the centromere of acrocentric chromosome as a marker;

(3) detecting chromosome copy number of the progeny DNA sample;

(4) determining genotypes of the target sites of both of the parents and the progeny according to the SNP site screened in step (2);

(5) analyzing haplotypes of both of the parents and the progeny based on the genotypes and pedigree relations of both of the parents and the progeny;

(6) analyzing the chromosomal Robertsonian translocation of the progeny according to the detecting result of chromosome copy number of the progeny, the karyotypes of both of the parents carrying chromosomal Robertsonian translocation and the haplotypes of both of the parents and the progeny.

Preferably, the method is useful for detecting human chromosomal translocation, particularly the translocation between any two chromosomes of Chromosome 13, Chromosome 14, Chromosome 15, Chromosome 21 and Chromosome 22.

In a specific embodiment, the parental DNA sample is the parental genomic DNA extracted from peripheral blood sample; the progeny DNA sample is selected from the group consisting of trophoblast cell whole genomic amplified products of embryo or fetus or genomic DNA extracted from abortion tissue, preferably blastocyst trophoblast cell whole genomic amplified product of embryo cultured in vitro.

In the above step (2), the screening criteria for SNP site are:

a. the site is within 10 Mb location in proximity to the centromere;

b. the site is collected in 1000 Genomes database or dbSNP database, preferably SNP of which the Minimum Allele Frequency (MAF) in target population is above 0.2;

c. the sequence in proximity to the SNP site is not homologous in human genome.

In a particular embodiment, the SNP sites comprise at least one SNP site, preferably at least 10 SNP sites, more preferably at least 50 SNP sites from every chromosome with translocation.

Preferably, the SNP site is selected from the group consisting of the following SNP sites or any combination thereof: chr13:19602195, chr13:19625370, chr13:19646283, chr13:19692745, chr13:19713634, chr13:19722801, chr13:19732341, chr13:19784913, chr13:19843135, chr13:19877547, chr13:19883672, chr13:19896927, chr13:19917509, chr13:20494558, chr13:20709214, chr13:20747500, chr13:20784332, chr13:20825315, chr13:20864572, chr13:20881859, chr13:20908501, chr13:20938952, chr13:20951425, chr13:20992333, chr13:21012562, chr13:21066214, chr13:21092894, chr13:21112935, chr13:21527671, chr13:21553971, chr13:21575458, chr13:21607760, chr13:21636136, chr13:21655335, chr13:21683681, chr13:21801501, chr13:22197910, chr13:22249975, chr13:22270788, chr13:22340074, chr13:22396664, chr13:22417688, chr13:22473749, chr13:22504691, chr13:22515844, chr13:22523279, chr13:22538311, chr13:22561684, chr13:22640820, chr13:22751654, chr13:22768476, chr13:22780737, chr13:22799808, chr13:22819627, chr13:22835073, chr13:22892864, chr13:22980146, chr14:19472367, chr14:20326905, chr14:20349694, chr14:20383719, chr14:20420387, chr14:20517369, chr14:20527044, chr14:20565487, chr14:20643775, chr14:20670979, chr14:20731036, chr14:20820537, chr14:20905141, chr14:20918598, chr14:21008574, chr14:21048774, chr14:21091127, chr14:21156472, chr14:21195967, chr14:21237137, chr14:21263166, chr14:21313819, chr14:21325985, chr14:21388266, chr14:21452591, chr14:21519915, chr14:21580671, chr14:21625262, chr14:21789092, chr14:21981923, chr14:22053817, chr14:22090208, chr14:22138437, chr14:22210067, chr14:22244363, chr14:22263821, chr14:22312275, chr14:22324460, chr14:22351977, chr14:22361282, chr14:22375208, chr14:22393124, chr14:22428984, chr14:22452496, chr14:22489611, chr14:22518909, chr14:22526966, chr14:22563283, chr14:22611651, chr14:22626948, chr14:22657897, chr14:22702039, chr14:22731529, chr14:22734948, chr14:22741160, chr14:22772663, chr14:22861948, chr14:22972955, chr15:20022190, chr15:20049922, chr15:20189367, chr15:20191854, chr15:20192138, chr15:20454253, chr15:20470948, chr15:20564500, chr15:20569241, chr15:20586537, chr15:20586966, chr15:20590656, chr15:20616721, chr15:20626721, chr15:20646773, chr15:20848359, chr15:20849625, chr15:20852181, chr15:20862156, chr15:20863958, chr15:21905797, chr15:21928397, chr15:21938184, chr15:21940649, chr15:22013203, chr15:22299435, chr15:22302182, chr15:22376163, chr15:22427155, chr15:22441959, chr15:22476195, chr15:22520388, chr15:22539952, chr15:22780065, chr15:22798800, chr15:22833272, chr15:22854479, chr15:22869870, chr15:22893990, chr15:22905599, chr15:22916637, chr15:22923869, chr15:22940961, chr15:22971662, chr15:23000363, chr15:23010627, chr15:23020535, chr15:23044004, chr15:23053813, chr15:23064687, chr15:23721906, chr15:23730971, chr15:23746510, chr15:23767135, chr15:23781336, chr15:23793989, chr15:23816231, chr15:23893430, chr15:23929209, chr15:24006330, chr15:24055535, chr15:24129894, chr15:24166516, chr15:24196094, chr15:24236177, chr15:24278723, chr15:24319610, chr15:24350583, chr15:24471640, chr15:24824114, chr15:24870875, chr15:24913763, chr15:24943341, chr15:24974072, chr21:14640496, chr21:14643423, chr21:14695894, chr21:14756515, chr21:14818516, chr21:14890028, chr21:15012159, chr21:15026905, chr21:15170049, chr21:15280687, chr21:15300595, chr21:15445542, chr21:15479041, chr21:15501432, chr21:15548748, chr21:15566056, chr21:15588845, chr21:15625530, chr21:15654618, chr21:15681452, chr21:15720558, chr21:15763305, chr21:15842397, chr21:15884516, chr21:15916232, chr21:15942816, chr21:15984234, chr21:16027704, chr21:16063848, chr21:16122140, chr21:16160041, chr21:16193235, chr21:16238898, chr21:16315932, chr21: 16353639, chr21:16384555, chr21: 16448591, chr21: 16578538, chr21: 16645724, chr21:16717265, chr21:16833315, chr21:16881542, chr21:17000938, chr21:17054831, chr21:17140290, chr21:17203891, chr21:17295288, chr21:17340912, chr21:17536247, chr21: 17640426, chr21: 17673255, chr21: 17713264, chr21: 17748703, chr21: 17781159, chr21: 17823782, chr21: 18003355, chr21: 18046650, chr21: 18114449, chr21:18151760, chr21: 18182817, chr21:18250262, chr21: 18337678, chr21: 18385770, chr21: 18420873, chr21: 18457287, chr21:18586049, chr21: 18650837, chr21: 18740886, chr21: 18858018, chr21:18936951, chr21:19002939, chr21:19064396, chr21:19139888, chr21:19254923, chr21: 19316600, chr22:16415691, chr22: 16554800, chr22: 16848067, chr22: 16850056, chr22: 16850858, chr22:16851557, chr22: 16852464, chr22: 16852792, chr22: 16853453, chr22: 16854397, chr22:16855647, chr22: 16856500, chr22: 16857432, chr22: 16858287, chr22: 16860547, chr22:16861742, chr22: 16867995, chr22: 17031697, chr22: 17271213, chr22:17293257, chr22:17409957, chr22:17442318, chr22:17469090, chr22:17525677, chr22: 17563259, chr22: 17587785, chr22: 17619511, chr22: 17656792, chr22:17682387, chr22: 17715164, chr22: 17739757, chr22: 17761489, chr22: 17785311, chr22: 17805528, chr22: 17841251, chr22: 17883346, chr22: 17916190, chr22: 17939998, chr22: 17981144, chr22: 18012297, chr22:18054406, chr22: 18212058, chr22: 18276198, chr22: 18354015, chr22: 18395978, chr22:18466951, chr22: 18531582, chr22: 18581715, chr22: 18628321, chr22:18894617, chr22:18966077, chr22:19004846, chr22:19025459, chr22:19098323, chr22: 19142652, chr22:19236590, chr22: 19362714, chr22: 19431588, chr22: 19536367, chr22: 19643965, chr22:19716983, chr22: 19759532, chr22: 19808057, chr22: 19859087, chr22:19909387, chr22:19972494, chr22:20016622, chr22:20051573, chr22:20081918, chr22:20135421, chr22:20175034, chr22:20216874, chr22:20278637, chr22:20745699, chr22:20787955, chr22:20855585, chr22:20881195, chr22:20924097, chr22:20983921, chr22:21028224. Most preferably, the SNP sites comprise all the above SNP sites from every chromosome with translocation.

In a particular embodiment, the process for detecting the chromosome copy number in above step (3) and step (4) is selected independently from the group consisting of sequencing process and chip process. The sequencing process is a high-throughput sequencing process, preferably Ion Torrent PGM or Illumina Miseq; the chip process is human whole genomic SNP chip, preferably Illumina whole genomic SNP chip.

Preferably, in the detecting method according to the invention, one of the parents has normal karyotype, the other carries Robertsonian translocation; the progeny comprises a subject that is Robertsonian translocation chromosome trisomic or monosomic.

Specifically, the determining criteria in step (6) are:

a. if a progeny is chromosome m trisomic, haplotype B in chromosome n from parental Robertsonian translocation carrier is translocation chromosome haplotype, and accordingly another haplotype b in chromosome n from parental Robertsonian translocation carrier is normal chromosome haplotype; and further by observing haplotype of other progeny, chromosome m haplotype A from parental Robertsonian translocation carrier linked with chromosome n haplotype B is translocation chromosome haplotype, and accordingly, another haplotype a in chromosome m from parental Robertsonian translocation carrier is normal chromosome haplotype; thereby it can be determined that the progeny carrying chromosome m haplotype A and chromosome n haplotype B is translocation carrier, and the progeny carrying chromosome m haplotype a and chromosome n haplotype b is normal karyotype;

b. if a progeny is chromosome m monosomic, haplotype B in chromosome n from parental Robertsonian translocation carrier is normal chromosome haplotype, and accordingly another haplotype b in chromosome n from parental Robertsonian translocation carrier is translocation chromosome haplotype; and further by observing haplotype of other progeny, chromosome m haplotype a from parental Robertsonian translocation carrier linked with chromosome n haplotype b is translocation chromosome haplotype, and accordingly another haplotype A in chromosome m from parental Robertsonian translocation carrier is normal chromosome haplotype; thereby it can be determined that the progeny carrying chromosome m haplotype A and chromosome n haplotype B is normal karyotype, and the progeny carrying chromosome m haplotype a and chromosome n haplotype b is translocation carrier;

wherein chromosome m and chromosome n are two chromosomes with Robertsonian translocation in the genome of parental Robertsonian translocation carrier, A and a are haplotypes from parental Robertsonian translocation carrier chromosome m, and B and b are haplotype from parental Robertsonian translocation carrier chromosome n.

Taking chromosomal translocation in Chromosome 13 and Chromosome 14 as an example,

+13 +14 −13 −14 translocation normal carrier embryo embryo embryo embryo embryo embryo Chr13 Aa Aa A — a A a Chr14 Bb B Bb b — B b

According to the above table, if Chromosome 13 is triploid or haploid, it can be inferred that B type in Robertsonian translocation carrier Chromosome 14 is translocation chromosome haplotype, b type is normal chromosome haplotype; according to other embryo carrying B type or b type, it can be further inferred that A type in Chromosome 13 is translocation chromosome haplotype, a type is normal chromosome haplotype;

if Chromosome 14 is triploid or haploid, it can be inferred that A type in Robertsonian translocation carrier Chromosome 13 is translocation chromosome haplotype, a type is normal chromosome haplotype; according to other embryo carrying A type or a type, it can be further inferred that B in Chromosome 14 is translocation chromosome haplotype, b is normal chromosome haplotype.

Therefore, carrier of Chromosome 13 a type and Chromosome 14 b type is normal embryo, and carrier of Chromosome 13 A type and Chromosome 14 B type is translocation carrier embryo.

The method according to the invention is particularly useful for preimplantation genetic diagnosis.

In the second aspect, provided is a primer composition for detecting chromosomal Robertsonian translocation, the primer composition is capable of amplifying the sequence of hypermutational SNP site in proximity to centromere of acrocentric chromosome. Preferably, the primer composition is for use in detecting human chromosomal Robertsonian translocation, particularly a translocation between any two of Chromosome 13, Chromosome 14, Chromosome 15, Chromosome 21 and Chromosome 22.

The screening criteria for SNP site are:

a. the site is within 10Mb location in proximity to the centromere;

b. the site is collected in 1000 Genomes database or dbSNP database, preferably SNP of which the Minimum Allele Frequency (MAF) in target population is above 0.2;

c. the sequence in proximity to the SNP site is not homologous in human genome.

In a specific embodiment, the SNP sites comprise at least one SNP site, preferably at least 10 SNP sites, more preferably at least 50 SNP sites from every chromosome with translocation.

Preferably, the SNP site is selected from the group consisting of the following SNP sites or any combination thereof: chr13:19602195, chr13:19625370, chr13:19646283, chr13:19692745, chr13:19713634, chr13:19722801, chr13:19732341, chr13:19784913, chr13:19843135, chr13:19877547, chr13:19883672, chr13:19896927, chr13:19917509, chr13:20494558, chr13:20709214, chr13:20747500, chr13:20784332, chr13:20825315, chr13:20864572, chr13:20881859, chr13:20908501, chr13:20938952, chr13:20951425, chr13:20992333, chr13:21012562, chr13:21066214, chr13:21092894, chr13:21112935, chr13:21527671, chr13:21553971, chr13:21575458, chr13:21607760, chr13:21636136, chr13:21655335, chr13:21683681, chr13:21801501, chr13:22197910, chr13:22249975, chr13:22270788, chr13:22340074, chr13:22396664, chr13:22417688, chr13:22473749, chr13:22504691, chr13:22515844, chr13:22523279, chr13:22538311, chr13:22561684, chr13:22640820, chr13:22751654, chr13:22768476, chr13:22780737, chr13:22799808, chr13:22819627, chr13:22835073, chr13:22892864, chr13:22980146, chr14: 19472367, chr14:20326905, chr14:20349694, chr14:20383719, chr14:20420387, chr14:20517369, chr14:20527044, chr14:20565487, chr14:20643775, chr14:20670979, chr14:20731036, chr14:20820537, chr14:20905141, chr14:20918598, chr14:21008574, chr14:21048774, chr14:21091127, chr14:21156472, chr14:21195967, chr14:21237137, chr14:21263166, chr14:21313819, chr14:21325985, chr14:21388266, chr14:21452591, chr14:21519915, chr14:21580671, chr14:21625262, chr14:21789092, chr14:21981923, chr14:22053817, chr14:22090208, chr14:22138437, chr14:22210067, chr14:22244363, chr14:22263821, chr14:22312275, chr14:22324460, chr14:22351977, chr14:22361282, chr14:22375208, chr14:22393124, chr14:22428984, chr14:22452496, chr14:22489611, chr14:22518909, chr14:22526966, chr14:22563283, chr14:22611651, chr14:22626948, chr14:22657897, chr14:22702039, chr14:22731529, chr14:22734948, chr14:22741160, chr14:22772663, chr14:22861948, chr14:22972955, chr15:20022190, chr15:20049922, chr15:20189367, chr15:20191854, chr15:20192138, chr15:20454253, chr15:20470948, chr15:20564500, chr15:20569241, chrl 5:20586537, chr15:20586966, chrl 5:20590656, chr15:20616721, chr15:20626721, chrl 5:20646773, chr15:20848359, chrl 5:20849625, chr15:20852181, chr15:20862156, chr15:20863958, chr15:21905797, chr15:21928397, chr15:21938184, chr15:21940649, chr15:22013203, chr15:22299435, chr15:22302182, chr15:22376163, chr15:22427155, chr15:22441959, chr15:22476195, chr15:22520388, chr15:22539952, chr15:22780065, chr15:22798800, chr15:22833272, chr15:22854479, chr15:22869870, chr15:22893990, chr15:22905599, chr15:22916637, chr15:22923869, chr15:22940961, chr15:22971662, chr15:23000363, chr15:23010627, chr15:23020535, chr15:23044004, chr15:23053813, chr15:23064687, chr15:23721906, chr15:23730971, chr15:23746510, chr15:23767135, chr15:23781336, chr15:23793989, chr15:23816231, chr15:23893430, chr15:23929209, chr15:24006330, chr15:24055535, chr15:24129894, chr15:24166516, chr15:24196094, chr15:24236177, chr15:24278723, chr15:24319610, chr15:24350583, chr15:24471640, chr15:24824114, chr15:24870875, chr15:24913763, chr15:24943341, chr15:24974072, chr21:14640496, chr21:14643423, chr21:14695894, chr21:14756515, chr21:14818516, chr21:14890028, chr21:15012159, chr21:15026905, chr21:15170049, chr21:15280687, chr21:15300595, chr21:15445542, chr21:15479041, chr21:15501432, chr21:15548748, chr21:15566056, chr21:15588845, chr21:15625530, chr21:15654618, chr21:15681452, chr21:15720558, chr21:15763305, chr21:15842397, chr21:15884516, chr21:15916232, chr21:15942816, chr21:15984234, chr21:16027704, chr21:16063848, chr21:16122140, chr21:16160041, chr21:16193235, chr21:16238898, chr21:16315932, chr21:16353639, chr21: 16384555, chr21:16448591, chr21:16578538, chr21:16645724, chr21:16717265, chr21:16833315, chr21:16881542, chr21:17000938, chr21:17054831, chr21:17140290, chr21:17203891, chr21:17295288, chr21:17340912, chr21:17536247, chr21:17640426, chr21:17673255, chr21:17713264, chr21:17748703, chr21:17781159, chr21:17823782, chr21:18003355, chr21:18046650, chr21:18114449, chr21:18151760, chr21:18182817, chr21:18250262, chr21:18337678, chr21:18385770, chr21:18420873, chr21:18457287, chr21:18586049, chr21:18650837, chr21:18740886, chr21:18858018, chr21:18936951, chr21:19002939, chr21:19064396, chr21:19139888, chr21:19254923, chr21:19316600, chr22:16415691, chr22:16554800, chr22:16848067, chr22:16850056, chr22: 16850858, chr22:16851557, chr22: 16852464, chr22: 16852792, chr22: 16853453, chr22: 16854397, chr22:16855647, chr22: 16856500, chr22: 16857432, chr22: 16858287, chr22: 16860547, chr22:16861742, chr22: 16867995, chr22: 17031697, chr22: 17271213, chr22: 17293257, chr22: 17409957, chr22: 17442318, chr22: 17469090, chr22: 17525677, chr22: 17563259, chr22: 17587785, chr22: 17619511, chr22: 17656792, chr22:17682387, chr22: 17715164, chr22: 17739757, chr22: 17761489, chr22: 17785311, chr22:17805528, chr22: 17841251, chr22: 17883346, chr22: 17916190, chr22: 17939998, chr22: 17981144, chr22: 18012297, chr22: 18054406, chr22: 18212058, chr22: 18276198, chr22: 18354015, chr22:18395978, chr22:18466951, chr22:18531582, chr22:18581715, chr22:18628321, chr22: 18894617, chr22: 18966077, chr22: 19004846, chr22: 19025459, chr22: 19098323, chr22: 19142652, chr22:19236590, chr22: 19362714, chr22: 19431588, chr22: 19536367, chr22: 19643965, chr22:19716983, chr22: 19759532, chr22: 19808057, chr22: 19859087, chr22: 19909387, chr22: 19972494, chr22:20016622, chr22:20051573, chr22:20081918, chr22:20135421, chr22:20175034, chr22:20216874, chr22:20278637, chr22:20745699, chr22:20787955, chr22:20855585, chr22:20881195, chr22:20924097, chr22:20983921, chr22:21028224. Most preferably, the SNP sites comprise all the above SNP sites from every chromosome with translocation

In a particularly preferable embodiment, the primer composition is selected from the group consisting of the following primer pair composed of the following forward primer and reverse primer, or any combination thereof:

(1) the primer for amplifying the SNP site in Chromosome 13, which is selected from the group consisting of following primer pairs: forward primer sequence SEQ ID NO:2n-1, reverse primer sequence SEQ ID NO:2n, n is a natural number selected from 1-57;

(2) the primer for amplifying the SNP site in Chromosome 14, which is selected from the group consisting of following primer pairs: forward primer sequence SEQ ID NO:2n-1, reverse primer sequence SEQ ID NO:2n; wherein n is a natural number selected from 58-115;

(3) the primer for amplifying the SNP site in Chromosome 15, which is selected from the group consisting of following primer pairs: forward primer sequence SEQ ID NO:2n-1, reverse primer sequence SEQ ID NO:2n; wherein n is a natural number selected from 116-189;

(4) the primer for amplifying the SNP site in Chromosome 21, which is selected from the group consisting of following primer pairs: forward primer sequence SEQ ID NO:2n-1, reverse primer sequence SEQ ID NO:2n; wherein n is a natural number selected from 190-264;

(5) the primer for amplifying the SNP site in Chromosome 22, which is selected from the group consisting of following primer pairs: forward primer sequence SEQ ID NO:2n-1, reverse primer sequence SEQ ID NO:2n wherein n is a natural number selected from 265-344.

Preferably, the primer composition comprises at least one pair of primers, preferably at least 10 pairs of primers, more preferably at least 50 pairs of primers for amplifying SNP site in every chromosome with translocation; and most preferably comprises all above primer pairs for amplifying SNP site in every chromosome with translocation.

Provided is also use of the primer composition according to the invention for the manufacture of a diagnostic reagent or kit for detecting chromosomal Robertsonian translocation.

The advantages of the invention are summarized as follows.

Applicability: The invention is capable of solving the detecting issue in embryo, fetus or abortion tissue Robertsonian translocation carrier, which is not possible for conventional methods.

Simplicity and Convenience: In the present invention, analysis of chromosomal translocation of various cells are based on cell chromosome copy number and haplotype without observation of the cells, thereby enable quick differentiation of normal and translocation carrying embryo.

Universality: Poly SNP is used in the present invention for analysis, which can be used in preimplantation genetic diagnosis, fetus diagnosis or abortion tissue diagnosis of different spouses.

High throughput: Based on high-throughput sequencing technology, the invention can be used to analyze chromosomal translocation and by different sequencing tags on each sample, a large number of samples can be analyzed at the same time.

Low cost: The detecting cost of chromosomal translocation of the invention is decreasing with the development of sequencing technology and decrease of sequencing cost.

High sensitivity: The invention can be used on the analysis for 3-5 cells. Accordingly, in addition to abortion tissue, trophoblast cell from cervix, the invention is particularly useful for preimplantation genetic diagnosis in test-tube baby technology.

Specificity: Hypermutational sites with Minimum Allele Frequency >0.2 of CHB (northern Han Chinese) and CHS (southern Han Chinese) within 10 Mb range in proximity to centromere location in chromosome in 1000 Genomes data are selected, wherein polymorphic sites with GC content >70% in polynucleotide (polyN) and upstream and downstream 50 bp sequences are excluded, and SNP mutant site which is uniquely aligned in human genome is selected as target location. Primers are designed by logging in https://www.ampliseq.com/ for submitting target site and location. These primers are highly specific.

Accuracy: According to change of translocation chromosome copy number and the sample carrying corresponding chromosome, the each derived chromosome haplotype in chromosome with translocation can be speculated, and higher accuracy can be achieved with mutual authentication.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the flow chart of the method for detecting chromosomal Robertsonian translocation according to the invention.

FIG. 2 shows the analysis chart of the method for detecting chromosomal Robertsonian translocation according to the invention for embryo translocation chromosome.

FIG. 3 shows chromosome pedigree haplotype pattern of Chromosome 13 pedigree haplotype in Example 1.

FIG. 4 shows chromosome pedigree haplotype pattern of Chromosome 14 pedigree haplotype in Example 1.

FIG. 5 shows chromosome pedigree haplotype pattern of Chromosome 13 pedigree haplotype in Example 3.

FIG. 6 shows chromosome pedigree haplotype pattern of Chromosome 14 pedigree haplotype in Example 2.

DETAILED DESCRIPTION

In context of the invention, reads refers to sequence fragments obtained in sequencing.

In context of the invention, single nucleotide polymorphism (SNP) refers to DNA sequence polymorphism resulted from single nucleotide variation at genomic level.

In context of the invention, haplotype refers to a combination of a group of single nucleotide polymorphism in a specific location of a chromosome which is associated with each other and are intended to inherit to progeny entirely. It is also known as haploid or haplotype.

In context of the invention, embryo genomic DNA is obtained by enriching genomic DNA in cells with whole genomic amplifying process, when an embryo develops to blastula stage where 3-5 periphery trophoblast cell are taken.

In context of the invention, enrichment of DNA molecule in target location is performed with multiplex PCR amplification. The specific principle and procedures can be found in the manufacture's instruction, where the DNA molecule is enriched into fragments with certain lengths and relatively concentrated. In a specific embodiment according to the invention, DNA fragments have the lengths of 125-275 bp.

In the present invention, according to hypermutational SNP sites in proximity to acrocentric location of human Chromosome 13, Chromosome 14, Chromosome 15, Chromosome 21 and Chromosome 22, 57, 58, 74, 75 and 80 pairs of sequence specific primers are designed respectively (primer sequences and relevant SNP site information can be found in Table 1-5). These primers are characterized in that (1) unique in target chromosome; (2) have the same annealing temperature.

In the present invention, SNP site is indicated with the chromosome where the site is located and the location at the chromosome (hg19 reference genome sequence as standard), for example, chr13:19602195 indicates that the SNP site is located at 19602195 nucleotide in Chromosome 13, hg19 reference genome sequence.

TABLE 1 Hypermutational SNP sites in proximity to acrocentric location of Chromosome 13 and detecting primers chromosome: forward  reverse No. location primer sequence (5′->3′) primer sequence (5′->3′)  1 chr13:19602195 TTCCTCATGAACCTATTGATT TCTGACCGACAAATACCAC ATGTTTCAGAA AATCTC (SEQ ID NO: 1) (SEQ ID NO: 2)  2 chr13:19625370 TGTTCCAGGGATGGATGGTC GGGTAGAAAACTGGCCAG T ATGATG (SEQ ID NO: 3) (SEQ ID NO: 4)  3 chr13:19646283 CCTCACAGGAGAGGTTGTTT GCCTGTTTTTGTCTCTGAA GC ATAAGAACTC (SEQ ID NO: 5) (SEQ ID NO: 6)  4 chr13:19692745 GCAGTCCAGGAATCCTCCTG ATCCCAAAACTATTTTCCTT TA CTCCTATCAC (SEQ ID NO: 7) (SEQ ID NO: 8)  5 chr13:19713634 GTCTGGTTGGTTAGAATGTT CACAGTGGAGCAGTTTAG CCATGA ACAGAG (SEQ ID NO: 9) (SEQ ID NO: 10)  6 chr13:19722801 AGTAACATGTTCCTTGTTTG CATGGCAGTGGTTTTGCAA CTCTGT TTG (SEQ ID NO: 11) (SEQ ID NO: 12)  7 chr13:19732341 CATCATAGGGTTTGCCAGAT TGGCAGAGTTAATGAATCT GGT GTGACAG (SEQ ID NO: 13) (SEQ ID NO: 14)  8 chr13:19784913 CAAAAGTGAGTGCTTGGAA TGCAATTCTATCAATGGCAT AGCT ACAATGTAAA (SEQ ID NO: 15) (SEQ ID NO: 16)  9 chr13:19843135 GAAGAAGGTGAGAGATTGC CAACAGAGTGAGTTGCGT CTGA TTCTTC (SEQ ID NO: 17) (SEQ ID NO: 18) 10 chr13:19877547 ACTCTACAGAGCATAGACTG TGTTAGTCATAGTGTTAGAT CTCA CTCCATGTGA (SEQ ID NO: 19) (SEQ ID NO: 20) 11 chr13:19883672 GGAAGGTGGGATAATAAGG CTGTATTAATGTGTGCACA AAAACAGT CTTTGCTTAG (SEQ ID NO: 21) (SEQ ID NO: 22) 12 chr13:19896927 GTTCATCATCATAAAAACAC GCCTCAGGTAATCCTTTATA ACTGAAGTGT GCAAGATAAG (SEQ ID NO: 23) (SEQ ID NO: 24) 13 chr13:19917509 TCATCAGGATCAGGTTCTAA GCATCAAGGTTTTAAGATT AATCTTACAC TCTAGGTTACG (SEQ ID NO: 25) (SEQ ID NO: 26) 14 chr13:20494558 CCTCTAGGACAGAGATGGA GATACAAAACTCTGCCTGC CAAACT TTACAC (SEQ ID NO: 27) (SEQ ID NO: 28) 15 chr13:20709214 CCATTTTCTGTCTCTTGCTCT GGAAGATGGAAAACTTCT AGGTAT GGAAAAGGAT (SEQ ID NO: 29) (SEQ ID NO: 30) 16 chr13:20747500 CAGTATCAACAGAAAACAA CTGCAACTATACCATTTTAC GCTGATTCAA ATTTCCACT (SEQ ID NO: 31) (SEQ ID NO: 32) 17 chr13:20784332 CTGCCATCTGAATGGTTTCA CTTTTTCTCCTGACTTTGTG AGG GCAT (SEQ ID NO: 33) (SEQ ID NO: 34) 18 chr13:20825315 GTTATGTATTTGTGATGCATT CTTTAACTGAAGGCAAAAA GGCCTT TTAGCAGACT (SEQ ID NO: 35) (SEQ ID NO: 36) 19 chr13:20864572 CAGTTACTTAGAACAGCAA AAGTGCATGACTTAGAGCC GATACTGAACA ACAA (SEQ ID NO: 37) (SEQ ID NO: 38) 20 chr13:20881859 CCAAGTGAATGGTGGTCTCA ATCTCCAAACATCTGCCAA CT CACT (SEQ ID NO: 39) (SEQ ID NO: 40) 21 chr13:20908501 GTACACACACTGCATCTCGT GGCACCAGGATAGTCACTG ATGT TAG (SEQ ID NO: 41) (SEQ ID NO: 42) 22 chr13:20938952 GGTGTCTGTATCCACTCCAA GTGCTCTTGATCTAACTGG TTCC AGCTT (SEQ ID NO: 43) (SEQ ID NO: 44) 23 chr13:20951425 AAAGCAAAACAACCATGCA GGGAAAAGCATCCCATCTT CCATTA TCC (SEQ ID NO: 45) (SEQ ID NO: 46) 24 chr13:20992333 TCCTCAGGGAGATCTCAGTT GTTAGATGTGTCTTGCTTC CTG CTCTTCTC (SEQ ID NO: 47) (SEQ ID NO: 48) 25 chr13:21012562 CATCTACAGATCCGGTAGGA GGAGAATCGTGAGGGTTA GTCT CTCTGA (SEQ ID NO: 49) (SEQ ID NO: 50) 26 chr13:21066214 ACCAAATATGTGTCTTGGCC GAGTGCTGTTGATGCTGAA AGTT TCG (SEQ ID NO: 51) (SEQ ID NO: 52) 27 chr13:21092894 CAGAAGCAATGGAGGGATT GTACTGTGAAAATACTGCA TTGG CATTCATTTCT (SEQ ID NO: 53) (SEQ ID NO: 54) 28 chr13:21112935 GCCTGGTGACAGAGTATGAT GAGGTCACAGCAATGTCA GAC GGAA (SEQ ID NO: 55) (SEQ ID NO: 56) 29 chr13:21527671 GCACCAAACAAGGACACAT TTTTTCCTTAATCTACCCAA AGTG ATACCAGGTA (SEQ ID NO: 57) (SEQ ID NO: 58) 30 chr13:21553971 AATGCTGACCAAAGATTCAT AGGAAATCTGTATGTTCAG GGGTA CATCTTAATGA (SEQ ID NO: 59) (SEQ ID NO: 60) 31 chr13:21575458 TTTTGTCACAATGGAGATAG CTCATTCCATTGATAGAGA GTGAACA AAATCACAGG (SEQ ID NO: 61) (SEQ ID NO: 62) 32 chr13:21607760 GTGACCTCAGACAGAGAGG AAGTGTAGTGGAACAAATT TAGA GTACTCTGT (SEQ ID NO: 63) (SEQ ID NO: 64) 33 chr13:21636136 GCCTCTCGGACCTATTTGAC GCTTCTGGCAAACGTCCTT TG CTTAAT (SEQ ID NO: 65) (SEQ ID NO: 66) 34 chr13:21655335 TTTGAAGACAAATTACCACT GTGATGATGAAGCCTTTCC TAAGGTTGTG CTTCA (SEQ ID NO: 67) (SEQ ID NO: 68) 35 chr13:21683681 GTGTCTGTTTTCTGAGTCCT CTCAGACTAGATAAAGGCT TCTTGT GGTAGAAAC (SEQ ID NO: 69) (SEQ ID NO: 70) 36 chr13:21801501 CTCAGAATCTCTTGGCTGTG TGAAGCCATGCTGATAGCA ACA TGT (SEQ ID NO: 71) (SEQ ID NO: 72) 37 chr13:22197910 GGCAGCCATTGAACTGGAA CAACATGGAAACCTCTCAC AAA ATTCAG (SEQ ID NO: 73) (SEQ ID NO: 74) 38 chr13:22249975 TCCTCACACTCAGTTTCTCT TCCCACCTAAGTTGTCACC AGAAAAGA TCT (SEQ ID NO: 75) (SEQ ID NO: 76) 39 chr13:22270788 GTATAGTTTATTTGAAGGCA GCCAATGTGTGATTTTTGT GACACTCCTT TTTAGACG (SEQ ID NO: 77) (SEQ ID NO: 78) 40 chr13:22340074 ATCAGTGAGTGCTTAAACTA GAATTAGATTCAGGCTATG TTGGATCAA CATTTCAGG (SEQ ID NO: 79) (SEQ ID NO: 80) 41 chr13:22396664 TACCAAACATAGCGTGGACC GAATGGCAACGGAGTTGTT TTTT TCC (SEQ ID NO: 81) (SEQ ID NO: 82) 42 chr13:22417688 ACAGATTTTATTCGAATATCC CATTTGCCAGGATTTCTGC GGTTGGAT TCA (SEQ ID NO: 83) (SEQ ID NO: 84) 43 chr13:22473749 GTAGGGCTCAGTGCTTTCTG GCATCCTCTGTTTGAGCTA TT AGGT (SEQ ID NO: 85) (SEQ ID NO: 86) 44 chr13:22504691 CATTTATTTAACAAGGGACA TAACTCCGCAGTCCTTAGA CAGACCAAA CCA (SEQ ID NO: 87) (SEQ ID NO: 88) 45 chr13:22515844 ACCATGGCATGGTCACTAAA GGGTCGCTCTGGTGTATAA AACT GC (SEQ ID NO: 89) (SEQ ID NO: 90) 46 chr13:22523279 TTTGCAAAGACTTCCTGCTG CCTCCAAAAATAGTCAGTA AATG GTTCCTACAAT (SEQ ID NO: 91) (SEQ ID NO: 92) 47 chr13:22538311 CCTTGGACTTTTAGCTTCTA ATTGCCTGATATACCGCAG GAACTGTA TTTATTTATCT (SEQ ID NO: 93) (SEQ ID NO: 94) 48 chr13:22561684 CCCAAAAATCTAAAGCCATC ACATCATATGACGTAAAAA ACCTT TTCCTACAGCA (SEQ ID NO: 95) (SEQ ID NO: 96) 49 chr13:22640820 CACCAGCATTAGTTTAGGCA TAACGAAACTGTGCACTTG AACA TTCCT (SEQ ID NO: 97) (SEQ ID NO: 98) 50 chr13:22751654 TTAAAGAGGTAGAATCTGTA TGGATGTTGGTTCCCTACC GGACTTGGT TCT (SEQ ID NO: 99) (SEQ ID NO: 100) 51 chr13:22768476 TTCTGTGTTGCAAGTGACAG TTGCTGCTGACAACAAAGA AAAAC TGTTTT (SEQ ID NO: 101) (SEQ ID NO: 102) 52 chr13:22780737 ACAGTAGGTTAAACAGGATA TGAGATTTAAGTGCAAATG GGAAAACG GATGCAAC (SEQ ID NO: 103) (SEQ ID NO: 104) 53 chr13:22799808 GAAAACTGGTCAGAGGCAG AGAGGATGATTCCCTATAC CTAA CTCTGAGTA (SEQ ID NO: 105) (SEQ ID NO: 106) 54 chr13:22819627 GAATGTAGGGACCAAATTCC ACAGACAGACAGACAGAT ATAGACA AGTATATGTGT (SEQ ID NO: 107) (SEQ ID NO: 108) 55 chr13:22835073 CTTAATGCAGATGAGGAACT TCATGAAACCACTGTTTGG AATGCAC ATAATCATCAT (SEQ ID NO: 109) (SEQ ID NO: 110) 56 chr13:22892864 GCTTAATGCAGTGGGTCCTA CACTGTAACACTTTAAATG AACT GGTGAATTGTT (SEQ ID NO: 111) (SEQ ID NO: 112) 57 chr13:22980146 CTCAGTTGCAGAGCCACAG GGTGTGCCAGAATCTAAGA ACT GAAATTCTC (SEQ ID NO: 113) (SEQ ID NO: 114)

TABLE 2 Hypermutational SNP sites in proximity to acrocentric location of Chromosome 14 and detecting primers chromosome: forward  reverse No. location primer sequence (5′->3′) primer sequence (5′->3′)  1 chr14:19472367 TGATTTGATGTTCTAACCAG GCCCTATACATGTTCAGGT CACTAAAACT GGAAAG (SEQ ID NO: 115) (SEQ ID NO: 116)  2 chr14:20326905 GGGCCTTTCCCAAATGCTAT GGGATATAAAGGTATTCAAT (SEQ ID NO: 117) AAAGGATAGCAT (SEQ ID NO: 118)  3 chr14:20349694 TCTTGGTTACACCATGAGGT TAGGTATTCATCTCCTTATA AAATATGC TTGGCAGTGT (SEQ ID NO: 119) (SEQ ID NO: 120)  4 chr14:20383719 GTCATTCTTTTTGCATTAGCT ATGCCAAAAGTACTTTGTC GGTTGA CATAAATGTG (SEQ ID NO: 121) (SEQ ID NO: 122)  5 chr14:20420387 CTTTTCATGTCTGACATCTGT TGCCTAAGGTAATGATAAA ACCACT CCGAATGTG (SEQ ID NO: 123) (SEQ ID NO: 124)  6 chr14:20517369 CAGGACTGGGAGAGAAATA GATATTTGCATCAGAATCA GGAGA GATGCTTTTGT (SEQ ID NO: 125) (SEQ ID NO: 126)  7 chr14:20527044 GCTATGGGTTTTGACAAATG ATAGCTTTGCAATAGCCTCT TGTTATGT AAGTGTT (SEQ ID NO: 127) (SEQ ID NO: 128)  8 chr14:20565487 GTTGTTGTTGTTTGTTGGTTT ACTTGAAAAGAGGCTAAT AGATTACCA GTTAGTGAACT (SEQ ID NO: 129) (SEQ ID NO: 130)  9 chr14:20643775 CGTGTTCACATTGCCCTCTA AAACACCTCAAGGCAGAA CA TGACT (SEQ ID NO: 131) (SEQ ID NO: 132) 10 chr14:20670979 GCTTCGATCTACCATCTMC AGACCATTAGAGCACCAG TGT CTTTTT (SEQ ID NO: 133) (SEQ ID NO: 134) 11 chr14:20731036 CTGCCATGGTTGATTAGCAG GAGATCTATTCGCTTGAGA GA TAGGAAGTG (SEQ ID NO: 135) (SEQ ID NO: 136) 12 chr14:20820537 TCTTTGGAGATTTTCTGTCTT AACAGAGCACAGTTTTGC GTAAGTCC CTCTA (SEQ ID NO: 137) (SEQ ID NO: 138) 13 chr14:20905141 TGGGACCCAGGAGCGAATAT TCCTCACAGCATGCAGATC AG AAG (SEQ ID NO: 139) (SEQ ID NO: 140) 14 chr14:20918598 TTTGTATTAAGGCCGTTCTTC GTAAGAAACAGGGAGGGT ATATGTGA GATTAGG (SEQ ID NO: 141) (SEQ ID NO: 142) 15 chr14:21008574 CTGATGTTATCAGGCTATTGC CAGGTCACAAGAAAGCTT AGAAC TGTGAG (SEQ ID NO: 143) (SEQ ID NO: 144) 16 chr14:21048774 GGAAACATGAGTTCCTCTTA GCAAAGTTTGAACTCTACT CAGTGA GGATTAGATGA (SEQ ID NO: 145) (SEQ ID NO: 146) 17 chr14:21091127 TGGGTACCCAGTGAGCTTAG CTCAGGGAGCACCGCATA AT AACG (SEQ ID NO: 147) (SEQ ID NO: 148) 18 chr14:21156472 ATGGAAGAAGGAGAAACCC GTCACAGAGTAAGATATGG AGGAAACATG TCTTCCAGTA (SEQ ID NO: 149) (SEQ ID NO: 150) 19 chr14:21195967 GCGTTGTGTGAGAATAGAG CAAATGTCCTTTTCGGATT GAACA CAATCAGG (SEQ ID NO: 151) (SEQ ID NO: 152) 20 chr14:21237137 ACATTTGACATCATTAGAAG GTAATCAATCAAGCACCAA TGCTCTCA GACTAAAAGAG (SEQ ID NO: 153) (SEQ ID NO: 154) 21 chr14:21263166 CCTTTATGCAAGATGGTGGT GTTTGGTGGCACAGCTGTA TTTTGA ATC (SEQ ID NO: 155) (SEQ ID NO: 156) 22 chr14:21313819 ATCCCTTGTAGCAACATAAA CAGAACAATGGCAGTATTT TGGATTGA CTGAAGC (SEQ ID NO: 157) (SEQ ID NO: 158) 23 chr14:21325985 CCACGCCCTCAAGAATGAA CAAGCACGAGTTGAGGAG ACA TAGTC (SEQ ID NO: 159) (SEQ ID NO: 160) 24 chr14:21388266 AGTCCAGGTGCCTTTAATGT ATGCTGACACCCAGAGTAC ACTG ATG (SEQ ID NO: 161) (SEQ ID NO: 162) 25 chr14:21452591 GCTGCTAAGAAATATCTTCT GGCCCTTAAATATGAGTTG TCCAGACT ATGGTACATTT (SEQ ID NO: 163) (SEQ ID NO: 164) 26 chr14:21519915 GATGAATCCAGGAGTGAGG AGCTTCAACCTTCAAACAG AACAA CCT (SEQ ID NO: 165) (SEQ ID NO: 166) 27 chr14:21580671 CAAAACAAAACCCATTCGAT TACTTGCAACAAAGAAGG CCAGA AGAATACTGG (SEQ ID NO: 167) (SEQ ID NO: 168) 28 chr14:21625262 AGTGAGTCTTTGTGAAAATG GTGTGTTATTCGCTTACTCT CCTTTTC CCTCA (SEQ ID NO: 169) (SEQ ID NO: 170) 29 chr14:21789092 TGCTTGAGGACACTTTTTGG GTAAAATGCCAGCAGCAG AAAAC AAACTAC (SEQ ID NO: 171) (SEQ ID NO: 172) 30 chr14:21981923 TTGTAGAAGGTACATGGTAC CACATACATCTTTCAATAGC CTGAGT TCCCTTCATA (SEQ ID NO: 173) (SEQ ID NO: 174) 31 chr14:22053817 TCAATAGCTTCTGGCTTCAA CATGAGAGATTCTTGGGAC TCTACTG CTGAAA (SEQ ID NO: 175) (SEQ ID NO: 176) 32 chr14:22090208 GGGAGCTTTCCTTCTCTATG GATGCTTCCATGTGGTCAC TTTCC AAAAG (SEQ ID NO: 177) (SEQ ID NO: 178) 33 chr14:22138437 GCGGGAATAGATGAAGATG GGTCCTGATGAGATTGACA CAGT ACTTCTTC (SEQ ID NO: 179) (SEQ ID NO: 180) 34 chr14:22210067 ATGAAACCATTAGCTCATCA CACACTGCTAATAAATTCA GGAATTAGTT AAATGGCAAAA (SEQ ID NO: 181) (SEQ ID NO: 182) 35 chr14:22244363 CATGAGATACACTACATACT CCCTAGCCACTTCTTGTCT CACCCAA GTG (SEQ ID NO: 183) (SEQ ID NO: 184) 36 chr14:22263821 TCTTATAAGTGAGAGACAAT CCTGTTCTTCTGAAAGAGG CTACAGGCAA CCTTTT (SEQ ID NO: 185) (SEQ ID NO: 186) 37 chr14:22312275 GGGCAAATGTGAGCCAAGA ACTTAGATGGTTTCTGTTA GTA ACAAGTGCT (SEQ ID NO: 187) (SEQ ID NO: 188) 38 chr14:22324460 TGTGCACACCATTGGCTGAT CACAAGGACACAAAGATG TA AGAACAATAAG (SEQ ID NO: 189) (SEQ ID NO: 190) 39 chr14:22351977 GTACAATAATTCCTAGAGCA CCAGAATCATTAGCTGTAT GAACACTCA GTAACGAGATG (SEQ ID NO: 191) (SEQ ID NO: 192) 40 chr14:22361282 TTTGAATCAGAAGCACATCC GCATAGAGATCATTGGACC TAGTAACATT CTAAAACTTAA (SEQ ID NO: 193) (SEQ ID NO: 194) 41 chr14:22375208 TTAGTGTAAAGGTAACAGAA TCATGATCACAGCTCAGTG TTTTCAGGCT TAACTTT (SEQ ID NO: 195) (SEQ ID NO: 196) 42 chr14:22393124 GCGGGAAATCAACATCACA CAGTCACATGTTTTTGAAT GAC CCAGTTCAAT (SEQ ID NO: 197) (SEQ ID NO: 198) 43 chr14:22428984 CCTTTAGTGAGGATTTGTCA TGAACTTCCTAAGCTCAGA GGTCTT GTTCCT (SEQ ID NO: 199) (SEQ ID NO: 200) 44 chr14:22452496 CCTATTAGTGAGCTATTGAAT ACCTCCTAACAAAGAAGA TTGGTTTGC CAAAAGACC (SEQ ID NO: 201) (SEQ ID NO: 202) 45 chr14:22489611 CCTAGGCTCCAATATTGAAA TAGTCCTGTGGTTAGCTTC AGTTTCAAAG CAGAA (SEQ ID NO: 203) (SEQ ID NO: 204) 46 chr14:22518909 AGGAAAGAGGTTTCATTGG GGGTAAATTTTCCCTTTGG CTGTT TGCT (SEQ ID NO: 205) (SEQ ID NO: 206) 47 chr14:22526966 TCAGAGAAGGCCTCTCTGA CCTTTCCCTTGAACAGTTA GAAAATA GAAGTCAC (SEQ ID NO: 207) (SEQ ID NO: 208) 48 chr14:22563283 GCAACTGGAGTCCTAGCCAT AAGAACTTTGTGCATGTCA TTG ATACCAC (SEQ ID NO: 209) (SEQ ID NO: 210) 49 chr14:22611651 GTGGCTGATGAGGCTGATGT GGCTAGGTCACATCAAACA AT ATTTACTTAAC (SEQ ID NO: 211) (SEQ ID NO: 212) 50 chr14:22626948 ATTCACAGGTTATCAAAGGA GTTCCCTGCTTAATCCAGT GAAGTATGAC ACAAAC (SEQ ID NO: 213) (SEQ ID NO: 214) 51 chr14:22657897 AGAAAACACCAAGACAAAA GCCCATAGGTACTGAAGCC CAGAGATTTG AAAG (SEQ ID NO: 215) (SEQ ID NO: 216) 52 chr14:22702039 ATGCCTTTGAACTGTTTGAA ATTCATTCGATATTAGTGTG ACTGAAT AGCATATGGT (SEQ ID NO: 217) (SEQ ID NO: 218) 53 chr14:22731529 TCAACACAATCTTAGATAGC GTAGCTTAATAACTGATAC AGTAGTGGTA AAGAGCCAAGA (SEQ ID NO: 219) (SEQ ID NO: 220) 54 chr14:22734948 TCCTGCTCTTTTCTAGGGTC CAGCTAATCTATTCATGGGT AGA CTATTGTTCA (SEQ ID NO: 221) (SEQ ID NO: 222) 55 chr14:22741160 CCTAGGTCCACTGCAGTAGA TCTAATCTAGCAGAACCTA AC GGGTTGT (SEQ ID NO: 223) (SEQ ID NO: 224) 56 chr14:22772663 TCCTCCTGCAGGAACTATGA GAATTATTTGTGACAGTTG CT CACTACATGTA (SEQ ID NO: 225) (SEQ ID NO: 226) 57 chr14:22861948 AACCTGGTGCTCAACTGTCT TTGAGCACTCTTCTTCTGT TATTAG TGAGAAAA (SEQ ID NO: 227) (SEQ ID NO: 228) 58 chr14:22972955 GCAACACAGGCAAACTAAT AATTCCAGATCCAATACAC CTTTGG CCTTCTTTT (SEQ ID NO: 229) (SEQ ID NO: 230)

TABLE 3 Hypermutational SNP sites in proximity to acrocentric location of Chromosome 15 and detecting primers chromosome: forward  reverse No. location primer sequence (5′->3′) primer sequence (5′->3′)  1 chr15:20022190 GAAGATATTTTCTTTCCAC AAGGAAGCCACTCAACTG ATTGTCCTCA CA (SEQ ID NO: 231) (SEQ ID NO: 232)  2 chr15:20049922 CGATGTCGCCTCTGTTCTG AAATACAATGCAAACTTG A TATTATCTCCATATG (SEQ ID NO: 233) (SEQ ID NO: 234)  3 chr15:20189367 TGGAGGTCTAATATATGGC CCCACATTCAGGATTGAG AATGTTCCT GATTATAATACA (SEQ ID NO: 235) (SEQ ID NO: 236)  4 chr15:20191854 ATAAAGAGACATCAGCATT GTTGTTGTACCAAATAAA CCGTATCAG CACAAAAACACT (SEQ ID NO: 237) (SEQ ID NO: 238)  5 chr15:20192138 GAGGCCTAATCCAAGGAG GCTTTGCAGGCCATAAATA AGAGAT TATACAACAAA (SEQ ID NO: 239) (SEQ ID NO: 240)  6 chr15:20454253 TCACAAGGCATTTGGGATC ACAGAGTGAGACCACACT CTC CCA (SEQ ID NO: 241) (SEQ ID NO: 242)  7 chr15:20470948 AAAAATCCTAACTGAAAC TTTATAAACATGCTGTTGT CCACTGT GTTGCAAAAA (SEQ ID NO: 243) (SEQ ID NO: 244)  8 chr15:20564500 GGGAGGGTAATCACACCA CTTCCATCTAAAGAAACA ATAGAG CCAATTTAGGAG (SEQ ID NO: 245) (SEQ ID NO: 246)  9 chr15:20569241 CTCTCTTTGGCCACATGAC TACCTTGTGTGCTAAGCTG CAT TTCTTATG (SEQ ID NO: 247) (SEQ ID NO: 248) 10 chr15:20586537 GAGTGGTGTGACTGTGGT CACTGACCTTCATAGGAG GAAT ACAACTG (SEQ ID NO: 249) (SEQ ID NO: 250) 11 chr15:20586966 TTGTGTCACAAACAAGAA AATTAAACATATCACAAAC GTCTTTTATTAAAT ATGCTTGGAATCTT (SEQ ID NO: 251) (SEQ ID NO: 252) 12 chr15:20590656 CAGAATCTGTAAACATGCT GTCTTTTGCAGCTCCTAG AGTTTGCAT AAGGT (SEQ ID NO: 253) (SEQ ID NO: 254) 13 chr15:20616721 AGGAGCATGCCATCAGGA CGAAAGGATTATGGAAGT AAAA GTCATGGTTA (SEQ ID NO: 255) (SEQ ID NO: 256) 14 chr15:20626721 GTTATGGCAGGACCATCTT AGCACACATTCTAAAGGA TCCA AATCACTCAA (SEQ ID NO: 257) (SEQ ID NO: 258) 15 chr15:20646773 CAGGACTCCACTACTCCCT CCCATGAGTGTGTCTGAAT GAA AACTTAGTATC (SEQ ID NO: 259) (SEQ ID NO: 260) 16 chr15:20848359 TATGGTAAGAATGTGGGTG AGAACTGAGTCCATCCCT ATGATTGAC GTAGTAC (SEQ ID NO: 261) (SEQ ID NO: 262) 17 chr15:20849625 CCTTCCTGTCTGACTCCTA ACTATGTTGAGATTTGACT AGCA GAATTTGCCT (SEQ ID NO: 263) (SEQ ID NO: 264) 18 chr15:20852181 CACAATTGACGGTCGCAA CAGCACTGGCTCCTACCT GTAC AAAA (SEQ ID NO: 265) (SEQ ID NO: 266) 19 chr15:20862156 GGCTACAATCCTTTAACTT TTTTGGTACCAAACAATG TCTCCAATCAA ATTAAGGGAGA (SEQ ID NO: 267) (SEQ ID NO: 268) 20 chr15:20863958 TAGAACAGCAACGTTTTG CGCCCTACTTTTATGTAGT TTTCTGC ACTTGAAGT (SEQ ID NO: 269) (SEQ ID NO: 270) 21 chr15:21905797 CAACCACAGCGAAACAGT TCTTCTGTGTCTAGAGGA GTC AGAGCTAC (SEQ ID NO: 271) (SEQ ID NO: 272) 22 chr15:21928397 GGTTTGATCAGGTGTGAA GAAGTTGTGCCAATAGGA GTTTACAG AAATACTACCT (SEQ ID NO: 273) (SEQ ID NO: 274) 23 chr15:21938184 CTTCCTTTCTCTGTGCCTG TCCCAGGAGCTCTGACCA AGAA A (SEQ ID NO: 275) (SEQ ID NO: 276) 24 chr15:21940649 CCAGTATAAAAAGGAACA CACAGTGAAAGTAAAACT CTTTACAGGGT AGGAAAATCTGC (SEQ ID NO: 277) (SEQ ID NO: 278) 25 chr15:22013203 GCGGCACCTGAGAATCCA GGTGAGGCTCCTTCAGTA TG GCT (SEQ ID NO: 279) (SEQ ID NO: 280) 26 chr15:22299435 AGGTAAACTTCCTCCTACA TCTCAAAATGCAAGCCCT TTCTTCTAAAA CTTGA (SEQ ID NO: 281) (SEQ ID NO: 282) 27 chr15:22302182 GGGCTGAACTTTAAGCTCT GTTTCAGTCAGTGTGAAT ATTCACT GAGACAGT (SEQ ID NO: 283) (SEQ ID NO: 284) 28 chr15:22376163 AACCAAGAATTTATTTTCA CTCAGAATTTTTAATGGCA TGCTTGCT TTACCATAAGGTT (SEQ ID NO: 285) (SEQ ID NO: 286) 29 chr15:22427155 GGTGTCTTTCTTGGGTCAA TGAAAATCAGCAGAGGTC GGT ATTGCT (SEQ ID NO: 287) (SEQ ID NO: 288) 30 chr15:22441959 CATGGTGAACACAAGCTT TTCAGTGCCGACAGCCAA GCTA GAGA (SEQ ID NO: 289) (SEQ ID NO: 290) 31 chr15:22476195 CACTCACATCCTGGTGTCT GCTGTGAATGCAATCTTG TGT GTAATCTG (SEQ ID NO: 291) (SEQ ID NO: 292) 32 chr15:22520388 CTTAGGAGTGAGCATGGT TACAATTAGGGTTAGTGTT CCTT AGGGTTAGGAT (SEQ ID NO: 293) (SEQ ID NO: 294) 33 chr15:22539952 ACATAGCACATGTGTGTGG GATGCATTACATGTTCCTT CAT ATGAACAGC (SEQ ID NO: 295) (SEQ ID NO: 296) 34 chr15:22780065 GTGTAGGTTGGTTGGACA TTCACTTCTCACGTGAGG GGAA ATCAAAG (SEQ ID NO: 297) (SEQ ID NO: 298) 35 chr15:22798800 GCTTTATCAATCACCCGTA TAGAGCTCACAACCTATG CAGTGA ATTTAACGC (SEQ ID NO: 299) (SEQ ID NO: 300) 36 chr15:22833272 CATGGATGTGTGTAGCGTG CCCGACGGTACGTTTTCTA ACT CTG (SEQ ID NO: 301) (SEQ ID NO: 302) 37 chr15:22854479 GTAGAGCTATTTGTACTCT CATATTGCTACCCTTCCAG TTTAGCCCA AACACTAA (SEQ ID NO: 303) (SEQ ID NO: 304) 38 chr15:22869870 GTCCTAGTTTCATTGGTGA CAGAACACAAACTGTGTA ATTTAAATGGC ACTAGGTTTG (SEQ ID NO: 305) (SEQ ID NO: 306) 39 chr15:22893990 CAGCGGTTTTCCAAGTGAT GACACCTGTCTTAACGCA GTC GGAA (SEQ ID NO: 307) (SEQ ID NO: 308) 40 chr15:22905599 ACTGGTCACATGGAATTG GGCCGACATATGTGAAGA ACTGT GGAA (SEQ ID NO: 309) (SEQ ID NO: 310) 41 chr15:22916637 CCCTGACTTGTTTGACTCC AGCAGCCAAGACTAACTT TTTG TAACCTG (SEQ ID NO: 311) (SEQ ID NO: 312) 42 chr15:22923869 GGCCAGCATGCTCTTTGTT ATGACGCTCAGCAGAGCA TTC CAGA (SEQ ID NO: 313) (SEQ ID NO: 314) 43 chr15:22940961 GCTTTCTCTCTGCACAGAT GAACCTGTCCCTGTCATTA GGA TGTGA (SEQ ID NO: 315) (SEQ ID NO: 316) 44 chr15:22971662 GGTGGTCTGCGACTGGAA AGAAAAATGGTAACATGA TTTA CAGTGGGAA (SEQ ID NO: 317) (SEQ ID NO: 318) 45 chr15:23000363 GGCTGGCTGTATGATCATC GATTCATTTTGGGAACGA GTA AAAGAAAGTTT (SEQ ID NO: 319) (SEQ ID NO: 320) 46 chr15:23010627 CAGGAGGCCCACAAAGAA TGCTCTTTGTGAGATTCTT TTCTA ATTCATCTGTT (SEQ ID NO: 321) (SEQ ID NO: 322) 47 chr15:23020535 AGCCAGGATAGAATGATG GGCTGGGATCTTTTGTCCA GAATGTG TGAT (SEQ ID NO: 323) (SEQ ID NO: 324) 48 chr15:23044004 TGCTTGGAAGCAGGAAGT GGTGTAAAGTTTGCATGA TCAG TTCCATGAA (SEQ ID NO: 325) (SEQ ID NO: 326) 49 chr15:23053813 ACATCTATAATCTCCAGGC CCTTAGGTTCAATGCTGA TTCTAACTTGT CCCTT (SEQ ID NO: 327) (SEQ ID NO: 328) 50 chr15:23064687 TACTGAGTCACCTCCATGG ATACACTGAGAGGGACAC GTT CAGACA (SEQ ID NO: 329) (SEQ ID NO: 330) 51 chr15:23721906 CGTCTAACCTGCAAAATG GCCGATGTTAATCTTTTTA CTAATATCTG CATCTCTCTC (SEQ ID NO: 331) (SEQ ID NO: 332) 52 chr15:23730971 CAATAACTGCACAAAGAA ACCATATTGCTGCTTTCTG GAGAGAAAATGG TAATTTTCATG (SEQ ID NO: 333) (SEQ ID NO: 334) 53 chr15:23746510 TGCCCTAATAAACAGACTC CATTCAGGCTGCCATAAC CAGAGA AAAATAGTAAAG (SEQ ID NO: 335) (SEQ ID NO: 336) 54 chr15:23767135 GCCTATTTCTCCAAATTCT ACCTTTCTTTATGTTATGAT TTCAGCCT TCAGCCAGAA (SEQ ID NO: 337) (SEQ ID NO: 338) 55 chr15:23781336 CAAACCTGGAAAGGAGTT GAATAAATCAGCTCAATA CTCTTGA GGCTGAATTGC (SEQ ID NO: 339) (SEQ ID NO: 340) 56 chr15:23793989 GTCCTGCCTCCTAGTTTTT TGGAGGTAAAGGCAAAC ATTGACT ACTGATC (SEQ ID NO: 341) (SEQ ID NO: 342) 57 chr15:23816231 CATGTGTCTCGATTTGATG CTAACTCTATCCCGCACTG GATATCCA AAGT (SEQ ID NO: 343) (SEQ ID NO: 344) 58 chr15:23893430 CAGACCAGTCAGACTCCA GCCTTTTCCATGTCTTTGA CATG CTCCT (SEQ ID NO: 345) (SEQ ID NO: 346) 59 chr15:23929209 CTCTGACAGCAGAGAGGA GATGTTTCATTTCTGGAGT AAGTG AGGACTGT (SEQ ID NO: 347) (SEQ ID NO: 348) 60 chr15:24006330 GGGATGTTGGCTTGTGTGT AACTTCCTTTCCTCTTTTT TTTT GCATTGTC (SEQ ID NO: 349) (SEQ ID NO: 350) 61 chr15:24055535 GTGAATGCCCTCTTCAATG GGTTACTTGAGGTGCAGA TAATGAAAT CTGA (SEQ ID NO: 351) (SEQ ID NO: 352) 62 chr15:24129894 AGACCTCTTGCATTTCCAT ACAGTTCATTTAAACAAG ATAGATTTTCA CATCAAGAAGAAAAA (SEQ ID NO: 353) (SEQ ID NO: 354) 63 chr15:24166516 TGGTTTGTTGGTTGATTGT ACATACATATTCTTTGCTAT TTGTGT CAGCTGACAA (SEQ ID NO: 355) (SEQ ID NO: 356) 64 chr15:24196094 AAACTTTGTTTAGTGGTTA ACTTGACAGAAAGTGTGA AACTAGGCAAT ACACCT (SEQ ID NO: 357) (SEQ ID NO: 358) 65 chr15:24236177 GGTTTGATACTTGTAGAGC CACAATGCAAGAGGAGAT CAGGTA GTCCT (SEQ ID NO: 359) (SEQ ID NO: 360) 66 chr15:24278723 TGCTTATTGTATGTGACTAT ATGCACAAGCATAAGAAA CACTTCCTTG CAGATGAAAAA (SEQ ID NO: 361) (SEQ ID NO: 362) 67 chr15:24319610 CCCATGCAGACAATCTTTG AGCTCCATCATATTCTCAT GTTG GAGACCT (SEQ ID NO: 363) (SEQ ID NO: 364) 68 chr15:24350583 AGGTTTTATGGTTTTTATG AACAAAACTATACATGTAT GTTTTTATGGTTT CCACAAACCTAAAT (SEQ ID NO: 365) (SEQ ID NO: 366) 69 chr15:24471640 ACCTGGGTGACAAGAAAG GTCTTGTCCCTTCAGAGTT AGAGA TACAGT (SEQ ID NO: 367) (SEQ ID NO: 368) 70 chr15:24824114 AAAAATAGAAAGAAGTGT CCTTCTGCATGCCCTGTAT CCCTCATCTGT ACC (SEQ ID NO: 369) (SEQ ID NO: 370) 71 chr15:24870875 TCTGTCATTTCTTGTTTGC GAAAGGAGCCATAAGGAT TTTTGAGG GTATTCCAATAT (SEQ ID NO: 371) (SEQ ID NO: 372) 72 chr15:24913763 AGAAGTTCAGGTGATTAC AAATGGCAGAGATAGAGC TTGGCATT AGAAGTG (SEQ ID NO: 373) (SEQ ID NO: 374) 73 chr15:24943341 TCTGCTGTATGTTTCCATTT GCTAGCATTAAAAGAAAG CTGATAGAG TACGTGTATCTG (SEQ ID NO: 375) (SEQ ID NO: 376) 74 chr15:24974072 ACCTCCATGATGCCTATGA TGCTGTGCACCAGCCAAT GCT TAATA (SEQ ID NO: 377) (SEQ ID NO: 378)

TABLE 4 Hypermutational SNP sites in proximity to acrocentric location of Chromosome 21 and detecting primers chromosome: forward  reverse No. location primer sequence (5′->3′) primer sequence (5′->3′)  1 chr21:14640496 CCTTCGGATGAAAATGAG GGAGCTTTCAATGTAATTT GATAAGATAATATGA CTACTAGAATTGG (SEQ ID NO: 379) (SEQ ID NO: 380)  2 chr21:14643423 ACTCTTTATCTCATTTATCT CCCTGTAACAGGGCTTAC GAAGAAAATGCAC TTCATT (SEQ ID NO: 381) (SEQ ID NO: 382)  3 chr21:14695894 CAGACTTGCATAGAGCCT CTTAGCCTCCAACCTACAT GTATGT CTTTCTC (SEQ ID NO: 383) (SEQ ID NO: 384)  4 chr21:14756515 TCTCTTAGCTTTCAATTTG ACCTTCCTGAAAGAAAAT GTGGAAATGA GGCCTT (SEQ ID NO: 385) (SEQ ID NO: 386)  5 chr21:14818516 CAAGGTGGTAACAGCCTA CCTGTATCTTCGCTTCTGT CCAA TCTTAACAT (SEQ ID NO: 387) (SEQ ID NO: 388)  6 chr21:14890028 GAGGATGGTAAGGATAGA CACCTTCTTTTCTCAACCA GGAAAGAAGA CCTTTG (SEQ ID NO: 389) (SEQ ID NO: 390)  7 chr21:15012159 GCAGATAGAAGTGGCTGA TGCAGTTGAATTAAGAAC ACAGA ATGGTTTTATCCTAT (SEQ ID NO: 391) (SEQ ID NO: 392)  8 chr21:15026905 CAAATCCCTTTACAGGTTA TCAATGGGTCTTAATAATC GTTTGTAAAATCAG AAATGACTCCA (SEQ ID NO: 393) (SEQ ID NO: 394)  9 chr21:15170049 CTTTTTCCACCTTGAATGC GGGTTTTCTGGAGAGATA ATGAGA GTGGTAG (SEQ ID NO: 395) (SEQ ID NO: 396) 10 chr21:15280687 CCTCATCACTGTCACCTTA GAAAGAGAAAGTGAGAA GAGTG GATGTGTTGAATG (SEQ ID NO: 397) (SEQ ID NO: 398) 11 chr21:15300595 ACACATGGTCTCATCCAGA CAAACAAACAAACAGAA AATCTATTTTT AGGAATAGGACTG (SEQ ID NO: 399) (SEQ ID NO: 400) 12 chr21:15445542 GACCTGCCTCGCTACCTTT GCTTGTACTCAGAATGCAT TA GTCTC (SEQ ID NO: 401) (SEQ ID NO: 402) 13 chr21:15479041 TGAGTTATTCTGGAAGACT CACACAACCTCATGCTATT GTTATCACCT TTACTTTTCT (SEQ ID NO: 403) (SEQ ID NO: 404) 14 chr21:15501432 CAGAGCCTTCCTATATCCT AACAGGCTGACCACGTCA TAACAACG TAATT (SEQ ID NO: 405) (SEQ ID NO: 406) 15 chr21:15548748 TGCAAAATTTGGCTTTTTA GCCATGGCTAGAAGTTAA AATCATCTGC TGTATAGTTTTG (SEQ ID NO: 407) (SEQ ID NO: 408) 16 chr21:15566056 CAATGCAAAATGTAACTAA CGCCTGTGGTTCTGTTCTT AAGTGAGCAGA GTA (SEQ ID NO: 409) (SEQ ID NO: 410) 17 chr21:15588845 GTTCCTTCAGGTACCGTCT GGAAGCCCTTAATCAGAA CTG ACGGG (SEQ ID NO: 411) (SEQ ID NO: 412) 18 chr21:15625530 CTGGCGGTATCTCATCCAT CCTCTGAGATACATTTCAA CAA GAAGGGTT (SEQ ID NO: 413) (SEQ ID NO: 414) 19 chr21:15654618 GGAAGAACCTATTGAATA GATCTTCATGTTGCCAAAT GGCTGAACA TTAATGGAGAA (SEQ ID NO: 415) (SEQ ID NO: 416) 20 chr21:15681452 ACAACCACAAAAAGAATT ATGACAAGTTGGCTATAG TCCTATGTTGTC CTTGTTACATTA (SEQ ID NO: 417) (SEQ ID NO: 418) 21 chr21:15720558 TGACCTAATATCAAATTTT CCTGGACAAAGAGGAGG TCAGGGCGTAT TTTAAGTG (SEQ ID NO: 419) (SEQ ID NO: 420) 22 chr21:15763305 AGCTATGGATATAGATCAA GCATCCTTCAAGACATGAT TGCATGGATTT AAAAGGACTAA (SEQ ID NO: 421) (SEQ ID NO: 422) 23 chr21:15842397 CCAACTTGCAATGAGATG GCACAGGGTTTTGCTACA GTTCTT ATTTTACTTAAA (SEQ ID NO: 423) (SEQ ID NO: 424) 24 chr21:15884516 AATTGATGCAAATGTGCTA GCTCCTCGGGATACTCCTC CATTTTTCAG AAA (SEQ ID NO: 425) (SEQ ID NO: 426) 25 chr21:15916232 GGTCACTTGAAATAGAAC ACAGAACCATCAAGAAAT CAATGACAGT TGGTCTGA (SEQ ID NO: 427) (SEQ ID NO: 428) 26 chr21:15942816 CACAAATGAAAGCCACCC CGACTGCACATTCCAAAT TTAATATTCA CTGTC (SEQ ID NO: 429) (SEQ ID NO: 430) 27 chr21:15984234 ACTGCAGGACCCTGATAG TTTTTAATGTAGTGTGCCA GATC AACTTATGGG (SEQ ID NO: 431) (SEQ ID NO: 432) 28 chr21:16027704 CTAGACCTTTCATAAGAGA ACCTAAAATTGATCTGTTG GAATTAGAAAATGC TTTTCTTGCATTA (SEQ ID NO: 433) (SEQ ID NO: 434) 29 chr21:16063848 AAGTTGGTTGCTGCAGTTT AGTTTTACAGTCACCTGC TCTG TGATAATTTGA (SEQ ID NO: 435) (SEQ ID NO: 436) 30 chr21:16122140 GGGAGGCTTTGATAAAAG CACATCATTTACTTTGAAC CCCA ACTTCTGTCT (SEQ ID NO: 437) (SEQ ID NO: 438) 31 chr21:16160041 TCTACATGGCCCATCAGAG GGGATGTACATGGACATAA GAA TATAAGCCATG (SEQ ID NO: 439) (SEQ ID NO: 440) 32 chr21:16193235 GCAATGGTTGAGGAAATT GCCAACTGATGTTCCTTAA TCAAAATAGC TGGCA (SEQ ID NO: 441) (SEQ ID NO: 442) 33 chr21:16238898 CTGATCCAATATTACTGGC AAGAAATTGATCACATCA ATCCTTCT CAGTTCTGGA (SEQ ID NO: 443) (SEQ ID NO: 444) 34 chr21:16315932 CTTGTTGGAGACAAAGTG AATGCTTCAGATTTAGGA AATAAGCC AGAATTAGGAGT (SEQ ID NO: 445) (SEQ ID NO: 446) 35 chr21:16353639 AGAGTCAATAAATCTGACT TTGGTTGTATGGATAGATG AGGTCCCAT GGCAAA (SEQ ID NO: 447) (SEQ ID NO: 448) 36 chr21:16384555 CATCCTTTTTCCTCCCAAT GGTCAAATAAGCTTGGGA CATTATCTTTG GATGTTG (SEQ ID NO: 449) (SEQ ID NO: 450) 37 chr21:16448591 CCTTTTCATAGGCATTGAA ATTGATGGTGCATTAGAG AAATCGTTTTC GAAAAAGATTTG (SEQ ID NO: 451) (SEQ ID NO: 452) 38 chr21:16578538 TCAGTTTCCTCCAGATATA GGCAGTCTTGCCTGGTTG CTCTATGGTC ATAT (SEQ ID NO: 453) (SEQ ID NO: 454) 39 chr21:16645724 TGAAGAAAATAAATGCAA CAGAGTAAGATGCCTTTTA TTGTGAATGCCT TTTGCTAAAGT (SEQ ID NO: 455) (SEQ ID NO: 456) 40 chr21:16717265 ACTCAGTTCTTTTTCCTTG ACACTGACTGATTGAATTT ACTTCTTCC GTGTGGA (SEQ ID NO: 457) (SEQ ID NO: 458) 41 chr21:16833315 GCAATTTTTCTTTCTGAAA CAAATGGGATGCTCATCTT GTCTGAAGCT CTCAATTTTT (SEQ ID NO: 459) (SEQ ID NO: 460) 42 chr21:16881542 TCAGCCAGGGAAATAATTT ATCCTCCATCCAAGTACCC CTCTTTTCT TGT (SEQ ID NO: 461) (SEQ ID NO: 462) 43 chr21:17000938 GTTATCTGAATCCAAATAC CCTCTCTCAATGGAAAATT CTACACACACA CAGATGAATTG (SEQ ID NO: 463) (SEQ ID NO: 464) 44 chr21:17054831 GATGGAGCAGCTTCCTGT TTTAGGTGCAATTGTAAGT GTAA CACAGGA (SEQ ID NO: 465) (SEQ ID NO: 466) 45 chr21:17140290 GTGAAGCAAGCAGGAAAT TTCTCCCTAAGGGAACCTA TGACTAAA TTCAAAATTTT (SEQ ID NO: 467) (SEQ ID NO: 468) 46 chr21:17203891 TGTGGGTTTCATGGAATGT TATGTACATTAAACAGTAT GGT GCACTGGCAT (SEQ ID NO: 469) (SEQ ID NO: 470) 47 chr21:17295288 TCATGGGACTCTTATTTCC CCCAGAGGTAGCCATGTG ATTGCAA ACTA (SEQ ID NO: 471) (SEQ ID NO: 472) 48 chr21:17340912 AAAAGACAAAGAAAGCA CTTCCACCTCTGAAACAT AATGTGGAACA AGTAAGCTAC (SEQ ID NO: 473) (SEQ ID NO: 474) 49 chr21:17536247 CCCTCAGACTTTTCTTGTA CTCAGTGAGTGAAGCCAT AATCCTATAGG ACGATA (SEQ ID NO: 475) (SEQ ID NO: 476) 50 chr21:17640426 GGGAGTAAATGGTCTTTAG AGGGCAGTGGAAGTAAA GGATCAT ACCTTTTT (SEQ ID NO: 477) (SEQ ID NO: 478) 51 chr21:17673255 TGTTATATCCCACATCTCAT GAAAGGCCAGGAGAAAA TTCTGAGAGA GGTCTT (SEQ ID NO: 479) (SEQ ID NO: 480) 52 chr21:17713264 TGATAAATTTTTCAGATCC TCCAAGTTCTTTTCTGTTC TTCACCACTCT ACCTTATTGAT (SEQ ID NO: 481) (SEQ ID NO: 482) 53 chr21:17748703 CTAGTCCTCCCAACTTGCT GTAACATTGAGCAGCAGC TGT AACA (SEQ ID NO: 483) (SEQ ID NO: 484) 54 chr21:17781159 ATCTGGCCAGATGAAAAT CCCTACTGCGTTTAACAA GGGATT CCAATC (SEQ ID NO: 485) (SEQ ID NO: 486) 55 chr21:17823782 TGGAGTTTATCACCAAGTC ACAGAAAGAGCTTGTAAT ATTTTATGTCC TCTTCCTTTCA (SEQ ID NO: 487) (SEQ ID NO: 488) 56 chr21:18003355 CTTCACAGAATGGGATAA CCTGACACATTCTGCATTC CTGCCT CCAT (SEQ ID NO: 489) (SEQ ID NO: 490) 57 chr21:18046650 ACTTTGTTCCCTGAATATA GTATCTTAGATATTCCCAG AAGAGCAAGT CTTTGTGCA (SEQ ID NO: 491) (SEQ ID NO: 492) 58 chr21:18114449 CGAAGGGCTTTGTCTAGAT GTGGATGAGAGGTGTGGA AAACATAACA TCTT (SEQ ID NO: 493) (SEQ ID NO: 494) 59 chr21:18151760 CCCACTGACCAAATCTGG GGACATTCTTTGACACTCT ACAT GAAATAGC (SEQ ID NO: 495) (SEQ ID NO: 496) 60 chr21:18182817 TTGTTTGTAGTGACTACAC CTGACAGAAAATTTTCCA AACTCAGT ACAGCTGTT (SEQ ID NO: 497) (SEQ ID NO: 498) 61 chr21:18250262 CCTGAAAAATATGTGGATC GAAAAGAGAAACATCAA AACCGAAAA GAGTCAGTTTTGG (SEQ ID NO: 499) (SEQ ID NO: 500) 62 chr21:18337678 GCTCATTCTCATTTTTGGC CTACAGAATACCACTCAG TTCCA CTGCAA (SEQ ID NO: 501) (SEQ ID NO: 502) 63 chr21:18385770 TGAGGAGGATACTTCTGTT CCCTTTCTTTCCTGAAAAT ATTCCATCA AAGTGCAG (SEQ ID NO: 503) (SEQ ID NO: 504) 64 chr21:18420873 CCCACCTCCAAATTGTATC AGTACTGGTTGTGCATTAT CCATT TAAATATGCAA (SEQ ID NO: 505) (SEQ ID NO: 506) 65 chr21:18457287 ACTGCACTCAATGAAACC GGATTTGCCTTTAATACTC ATCTGA AGGGATACAAT (SEQ ID NO: 507) (SEQ ID NO: 508) 66 chr21:18586049 ACCATGTTTTAACTCTTCT GTCAAACCAGCTTAAGTT TTTAAAGCTGT TAAAAAGCCATA (SEQ ID NO: 509) (SEQ ID NO: 510) 67 chr21:18650837 GAGCATGTTTCCTGAACAA TTTTTGTTGTTGTAGATTG TCATTGG TTGTTGCTG (SEQ ID NO: 511) (SEQ ID NO: 512) 68 chr21:18740886 AGATGTATTACCTTCATAG TGTGGTTCCTCAGCTGTC TCTCTGGGTTT AAATC (SEQ ID NO: 513) (SEQ ID NO: 514) 69 chr21:18858018 CAACCAATCAAGCAGCAG GGTGCAGGGACAGCCTTT TGAAT TTAT (SEQ ID NO: 515) (SEQ ID NO: 516) 70 chr21:18936951 TGGTTTGTTGGCTTCACAG GCTGAAAACACTACAGAT ATGT TTAAGTTTGCTG (SEQ ID NO: 517) (SEQ ID NO: 518) 71 chr21:19002939 TCCTGTCATTTCACTCCTA AAGAAGGAAGAAGGAAG CATACTTCA TCTTCTCTACA (SEQ ID NO: 519) (SEQ ID NO: 520) 72 chr21:19064396 CGGTGCATTAGGAAGCCA CACATGAATATCCAAATGT AGTT CCAATTGTCAT (SEQ ID NO: 521) (SEQ ID NO: 522) 73 chr21:19139888 GCCCTACCTCCTAATACAA CTTTTCAGGTGCACCGTC CCATATC TTTG (SEQ ID NO: 523) (SEQ ID NO: 524) 74 chr21:19254923 GCCTGGCCTCTGTTTGTCT GTACTCTTATTAATGCCTC TTTATAA CTTTGGTCA (SEQ ID NO: 525) (SEQ ID NO: 526) 75 chr21:19316600 CTGCTTGACTTTAGAAGGA GGCTGGTGTAGAGCCACT TGATGGA TAAC (SEQ ID NO: 527) (SEQ ID NO: 528)

TABLE 5 Hypermutational SNP sites in proximity to acrocentric location of Chromosome 22 and detecting primers chromosome: forward  reverse No. location primer sequence (5′->3′) primer sequence (5′->3′)  1 chr22:16415691 CAGCCATTTTTCACGTTGC CCCTGGAGAAAGCAGTTC AGT TTTAC (SEQ ID NO: 529) (SEQ ID NO: 530)  2 chr22:16554800 CTAATTGAGGGACAGACA GCTACTGGATTCTGAGTG CTCACA TGTGTTC (SEQ ID NO: 531) (SEQ ID NO: 532)  3 chr22:16848067 TCGAATGGAGTTGAATGG CATTTGTGTCCATTCTATG AATTATCAAAGA ATTCCATTTCA (SEQ ID NO: 533) (SEQ ID NO: 534)  4 chr22:16850056 GAATAATCAAATGGAGAC CTATTCGGTGATTACATTC GAATGGAATCC GATTGCATTT (SEQ ID NO: 535) (SEQ ID NO: 536)  5 chr22:16850858 AGAATCGAATGGAATAATC ATTTTTCCATTTGATACCA ATCGAATGTACTA TTCGATGATGATTC (SEQ ID NO: 537) (SEQ ID NO: 538)  6 chr22:16851557 CTAAGGGAATAATCGAAC TCGATTTCTCCATTTAATT GGACTCAA CCATTCGATGA (SEQ ID NO: 539) (SEQ ID NO: 540)  7 chr22:16852464 TGGAATCATCGAATGTCAT TAGTCTATTCGATGATTCC TGAATGGA ATTCGAGTCTA (SEQ ID NO: 541) (SEQ ID NO: 542)  8 chr22:16852792 CGAAAGGAATCATCTTCA CCATTCGATTCCAAAAGAT AAAGGAAGTG GATTCCCT (SEQ ID NO: 543) (SEQ ID NO: 544)  9 chr22:16853453 AATGGAATCATCATGAATG CCATTCGAGTCCATTCAAC GAATCAAAAGG AATGATTG (SEQ ID NO: 545) (SEQ ID NO: 546) 10 chr22:16854397 GAAATCAAATGGAATCAAT GATTCAATTCGATGATGAT GAATGGACTCA TTGATTGGAGT (SEQ ID NO: 547) (SEQ ID NO: 548) 11 chr22:16855647 CACGAATGGAATCATCACT CGATTCCATTTGATTAGGA GAATGAAG TTCAATTCCAG (SEQ ID NO: 549) (SEQ ID NO: 550) 12 chr22:16856500 AGAATCGAACCGAATGGA GGTGATTACATTGAATTCC ATCATCTT TTTCGATGATG (SEQ ID NO: 551) (SEQ ID NO: 552) 13 chr22:16857432 TGGAATCATCATCAAATAG TCAATTCCATTTGATAATTA TATCGAAAGGAATC TTCCATTCGATTC (SEQ ID NO: 553) (SEQ ID NO: 554) 14 chr22:16858287 AAATGTAATCCAATGGAAT CATTCGATTCCTTTCGAGG CCTCGACT ATTTTATTCA (SEQ ID NO: 555) (SEQ ID NO: 556) 15 chr22:16860547 AATCATTGAATGGAATCAA TTGGTGACTCCATTTGAC ACAGAATCGTC ACCAT (SEQ ID NO: 557) (SEQ ID NO: 558) 16 chr22:16861742 TCGAATGGAATCATCCAAT CTCCGATCCATGATTCCAT GAAGTCTAATG TCGATT (SEQ ID NO: 559) (SEQ ID NO: 560) 17 chr22:16867995 GCTGGCCACGGCTTTTTAC CGGGTGTGCAAAAATCCG (SEQ ID NO: 561) C (SEQ ID NO: 562) 18 chr22:17031697 CCACTAAACAGAGATTCT GCGTCTCAACTTTATGTAC GGATTCAAGG CTTAGGTAAAC (SEQ ID NO: 563) (SEQ ID NO: 564) 19 chr22:17271213 CACTGCACTAACCCTAGG TTTTTCTGCGTGGAAGCTT CAATA TCTAG (SEQ ID NO: 565) (SEQ ID NO: 566) 20 chr22:17293257 CACTTCCTCCAGTTTTTCC GCAAGTGTGTACTTTAAA TACATAGA ATGGCTAATGTT (SEQ ID NO: 567) (SEQ ID NO: 568) 21 chr22:17409957 CTTGTTTCCTTGTCATTATC ACAGAAGTCAGTGAGGA TTAGGAGTCA GTGTGA (SEQ ID NO: 569) (SEQ ID NO: 570) 22 chr22:17442318 TTTTTGGAGGAGGAAAGT CACATTGGACTCAAACTT CTGTTCA GGCTATG (SEQ ID NO: 571) (SEQ ID NO: 572) 23 chr22:17469090 CTACCTTGCATGTTCTGCT CCTCTAGCAGCCCACTTTA CTCT GGA (SEQ ID NO: 573) (SEQ ID NO: 574) 24 chr22:17525677 CTTACAGCAGGGAACAAA CGGGTTCCAATCTGGGTT GTTCAC TCAA (SEQ ID NO: 575) (SEQ ID NO: 576) 25 chr22:17563259 CCTGGGCACTAGACATAG CACTGTGCCCAGTCAAAT CAAC ATTTTTGA (SEQ ID NO: 577) (SEQ ID NO: 578) 26 chr22:17587785 CGTCATCTGGGAAGCTGTT GTGCAGGACACTTCGGAA TCC AACAGAAG (SEQ ID NO: 579) (SEQ ID NO: 580) 27 chr22:17619511 AAATCAATGTTGCAAAGC AGCATCCTCACTTACCAGT AAAGGGA ATGC (SEQ ID NO: 581) (SEQ ID NO: 582) 28 chr22:17656792 AAAATCACCATTTGATGGT CATTATTTCTGCCATACTT TGTATTTTCCA CGTTGGT (SEQ ID NO: 583) (SEQ ID NO: 584) 29 chr22:17682387 CAGCATTCTGACTTTCTTG AGAGAGTCTGCTCGTTCA TTTACACA TTTACATATTG (SEQ ID NO: 585) (SEQ ID NO: 586) 30 chr22:17715164 GGAGACAGGTTCACATGA GTGAAAGCAGGAGCTTCT AAAAGTT TGGA (SEQ ID NO: 587) (SEQ ID NO: 588) 31 chr22:17739757 CCCAAGGAAAACGGGTTT CACACATGTGCATGCTACT ATGG GAA (SEQ ID NO: 589) (SEQ ID NO: 590) 32 chr22:17761489 AAGAAACTTGGTTTCATTT AGTAATAAGCAACCCAAA TCCATTAAGGT CTTAGCCA (SEQ ID NO: 591) (SEQ ID NO: 592) 33 chr22:17785311 TGGCTCTGACTTCATGATC GGCTTGGAAGCATAAGTA ATGG GGATGTAG (SEQ ID NO: 593) (SEQ ID NO: 594) 34 chr22:17805528 CCAGCCTGCCAACACAAT CCATTAACTGTATCTATGC ATTG TTGTCTGTCT (SEQ ID NO: 595) (SEQ ID NO: 596) 35 chr22:17841251 TATGGAAATCACTGAAAA GTTTTGTTTGTTTTTCAAA GTGGAGAAACT TGGTGTCTCA (SEQ ID NO: 597) (SEQ ID NO: 598) 36 chr22:17883346 TTGGAAATTCTTTCAGGTT AAAACAAGTTTGCCAGTT GACTCCTT TCTTTAGAAGTT (SEQ ID NO: 599) (SEQ ID NO: 600) 37 chr22:17916190 GGGAAAAGGATTTTGTATT GGCAAGACCATCTAATGG TCATGTCATCC CACT (SEQ ID NO: 601) (SEQ ID NO: 602) 38 chr22:17939998 CCCGGTATCCTTATGTCCC GGATGGAACTAAAGGCCA AGT GCTAA (SEQ ID NO: 603) (SEQ ID NO: 604) 39 chr22:17981144 TTTTTCCTTTAGAGCTGTG GCAAAGTGTATAACGCATT TGTGTAAG AGGATGAAGTA (SEQ ID NO: 605) (SEQ ID NO: 606) 40 chr22:18012297 GTGGCCTTTCCAGCTTCTA TTTCAAGTCATACACCAC GTAG CTCAAATATGT (SEQ ID NO: 607) (SEQ ID NO: 608) 41 chr22:18054406 CTGACATGGTGTCATACAC ACAGCATGTGCCCTCCTA CTATGG AAAG (SEQ ID NO: 609) (SEQ ID NO: 610) 42 chr22:18212058 AATACTTTACCTACCCTGA ACATCACTCCCTAAATGG TCACCAAAAC GACACTA (SEQ ID NO: 611) (SEQ ID NO: 612) 43 chr22:18276198 CCACATGCCTTTCAACGAC GGGCTTTAAGTCTCAGAT AAT CCATCA (SEQ ID NO: 613) (SEQ ID NO: 614) 44 chr22:18354015 TTCTATCAATGTCAAGGTC GCAACATGTGCTGCCAGT TTTATGCCA TTTT (SEQ ID NO: 615) (SEQ ID NO: 616) 45 chr22:18395978 ATGGCTTTTACTAGGTGAA AGAGACATTTAAATAATGT GGTAATTTTCT CCTTGCAGAGT (SEQ ID NO: 617) (SEQ ID NO: 618) 46 chr22:18466951 GGCAAAACGCAAATCATC GGACCAGGGAAATCCTGA GTGA GTCT (SEQ ID NO: 619) (SEQ ID NO: 620) 47 chr22:18531582 TGACCCTCTTAGAACCCAT AGGAGAGGCTTTTAAAAT TCCT CGCTATGATT (SEQ ID NO: 621) (SEQ ID NO: 622) 48 chr22:18581715 AGATGCATGAAATGGCTAC GTTGATTTGTGGTTGCCTA ATAGATATGG TGTAATTGTAT (SEQ ID NO: 623) (SEQ ID NO: 624) 49 chr22:18628321 GGCCCACACTTTCTTTTGA GGGATTTGGGAGAAATGC AACA AATGTC (SEQ ID NO: 625) (SEQ ID NO: 626) 50 chr22:18894617 CGCCTTTTTGGTGACCAAT GCTGTCAACAGACCTCCA GTG ACTA (SEQ ID NO: 627) (SEQ ID NO: 628) 51 chr22:18966077 CTGTGCTCTAAAGCCAGA CTCTGTACACTGCCACATA CTGA CCA (SEQ ID NO: 629) (SEQ ID NO: 630) 52 chr22:19004846 GCTGTATACGACAAAGCC TTTTTCTCTTGCATATTTGT ATAGCT GTGATGTCT (SEQ ID NO: 631) (SEQ ID NO: 632) 53 chr22:19025459 CTGTGTCAGGCAACAAAA CCCATTTGTGCCATAAAG CCAC GGTT (SEQ ID NO: 633) (SEQ ID NO: 634) 54 chr22:19098323 CAGAAGGAGAAAGACAA CCTCATTTTACAGATAGAG GGAGTAGATG AAACAGAGCTT (SEQ ID NO: 635) (SEQ ID NO: 636) 55 chr22:19142652 CACCAGGCCCTCATCTCTT ATGAGGGCAAAGACTCCC AAC AAAC (SEQ ID NO: 637) (SEQ ID NO: 638) 56 chr22:19236590 GCCTGGCCCTTCTAAGAG CATGATCATTTGAACTTAT TTTTA TCCTCCAGTCT (SEQ ID NO: 639) (SEQ ID NO: 640) 57 chr22:19362714 CCTCTCAGGAGGCAGAGA GGTAGAGAGCTGCAATAA ACTT AAATGATGTAAG (SEQ ID NO: 641) (SEQ ID NO: 642) 58 chr22:19431588 CAGGCAGTTTCCCAAGGA GATACCCTGACACTGCGA CTAG TGTT (SEQ ID NO: 643) (SEQ ID NO: 644) 59 chr22:19536367 CGTGAGTCTGGGTGACAG GGGAGCACATCAGCATTG T TATCA (SEQ ID NO: 645) (SEQ ID NO: 646) 60 chr22:19643965 ATACCAAGAAACCCAAGA GGAAGCTTCTCCAAGGTA AAGAAAGGGTGA AGCT (SEQ ID NO: 647) (SEQ ID NO: 648) 61 chr22:19716983 CACCTCCAGAGGTCCTAG CACTTTCTCCTGGGCTGTA TGTA CAT (SEQ ID NO: 649) (SEQ ID NO: 650) 62 chr22:19759532 GGGTCTCAGCCTAACTGA GGGAGGAGTGTCCTGAGA AGGA AATGAT (SEQ ID NO: 651) (SEQ ID NO: 652) 63 chr22:19808057 CATGAAGGGTCCAGTGGA GGAGTCAGGAACTCTCTG GAT ATGGA (SEQ ID NO: 653) (SEQ ID NO: 654) 64 chr22:19859087 CGCCAGCGTGTACCTCTTT GGCACGGGAGCATCCTAT (SEQ ID NO: 655) G (SEQ ID NO: 656) 65 chr22:19909387 CCAGAGGTCATGCACAGA TATGCTTTTCCCAGTCCAG AGAC ATTCTG (SEQ ID NO: 657) (SEQ ID NO: 658) 66 chr22:19972494 ACCGTGTATCGGAGACCC CAGCCAACTCCTGTTGAG TTAT AGTC (SEQ ID NO: 659) (SEQ ID NO: 660) 67 chr22:20016622 CTGAGAGCCAGGGAAGCA TCACTCCATGCGGTGTCTA GATC AAAA (SEQ ID NO: 661) (SEQ ID NO: 662) 68 chr22:20051573 TGCAGGGTGTCATTTCCTT GAAGCAAACATTCAGCAC CAG ACTCT (SEQ ID NO: 663) (SEQ ID NO: 664) 69 chr22:20081918 CTATAGACAGCTTGGCTCC GGCACGTGAGAAAACAG ATGT CTTTG (SEQ ID NO: 665) (SEQ ID NO: 666) 70 chr22:20135421 GCCTTAACTTCCTGGAGCC CCCAAATGGGAGATGTAG A AGCC (SEQ ID NO: 667) (SEQ ID NO: 668) 71 chr22:20175034 GGGATGCATTGCAGTAAA CCCATTCCCTGTCACTGTT GAGC ATCA (SEQ ID NO: 669) (SEQ ID NO: 670) 72 chr22:20216874 GGCTACAGAGACATGAAC GGCTCATGGCATGAACTT CCAA GCTA (SEQ ID NO: 671) (SEQ ID NO: 672) 73 chr22:20278637 GGGACAGTAGACAAGCTT GTCGAGATGCTTAATAGA ATCCCT GCCACAT (SEQ ID NO: 673) (SEQ ID NO: 674) 74 chr22:20745699 AGAAAGACTCAGACCTGA ACCTGTGCACAGGTGACT CCTGTT CTAT (SEQ ID NO: 675) (SEQ ID NO: 676) 75 chr22:20787955 GGATAGACTGGCCCTACA GTGGAAAATACACTGGAC CCAT CAATTCC (SEQ ID NO: 677) (SEQ ID NO: 678) 76 chr22:20855585 AAAAAGAAGATGGCGTAA GCAGAAGCAAATGGAAA TCAGAAGTTG GGAGAAATC (SEQ ID NO: 679) (SEQ ID NO: 680) 77 chr22:20881195 TATCTTCAGGCTCAGTGCT GAAGCTGGCAGAATGAGG AGTGA GAAT (SEQ ID NO: 681) (SEQ ID NO: 682) 78 chr22:20924097 GGGTGCTCAGTAATGTCCT CAGAGACCTCCAACCTCA TCA TCAC (SEQ ID NO: 683) (SEQ ID NO: 684) 79 chr22:20983921 TATGAGCAGGTCATGGCA CACTTAAATGTGAACAAA GGTA GTGTCCATTGA (SEQ ID NO: 685) (SEQ ID NO: 686) 80 chr22:21028224 TCAGGGAAGTATAAAAGT CCTCAGTTGCTCATCACTG GACAGGGAT TCAT (SEQ ID NO: 687) (SEQ ID NO: 688)

In the present invention, the sequencing process used may be high-throughput sequencing technology. The lengths of DNA fragments are 125-275 bp. In a specific embodiment according to the invention, the sequencing platform is Ion Torrent PGM and DNA sequence molecules with DNA lengths of 125-275 bp are obtained.

In the present invention, the sequencing depth may be 3003000×, i.e. each specific PCR amplified product is sequenced for 3003000 time. For example, in a specific embodiment of the invention, the sequencing depth is 1000, i.e. the specific PCR amplified product is sequenced for 1000 times.

In the present invention, when the DNA molecules for detection are from various samples, each sample can be provided with a different barcode so as to be differentiated during sequencing process (Micah Hamady, Jeffrey J Walker, J Kirk Harris et al. Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nature Methods, 2008, 5(3)), enabling sequencing of various samples at the same time.

In the present invention, the genome reference sequence may be obtained from public database. For example, the human genome sequence may be human genome reference sequence in NCBI or ucsc database.

In the present invention, the sequence alignment may be done with any sequence alignment program, for example, Torrent Mapping Alignment Program (TMAP) and BWA (Burrow-Wheeler-Aligner) available to a person skilled in the art can be used to perform alignment and the reads is aligned with reference genome sequence to give the location of the reads at the reference genome sequence.

In the present invention, Torrent_Server_4.0_VM software is used on raw data generated by PGM sequencer to remove linker sequence and Tmap software is used for alignment to human hg19 reference genome, and haplotype SNP covering multiple and genotype are analyzed finally.

In a particular embodiment according to the invention, the detecting method comprises the steps of:

DNA extracting and sequencing: After extracting cell DNA with MDA whole genomic amplifying process (Qiagen Kit), the library is constructed according to Ion AmpliSeq™Library Kits 2.0 standard procedures. During this period, embryo MDA whole genomic amplification is amplified to DNA molecules concentrated as about 125˜275 bp through multiplex PCR. Two ends are added with linkers for sequencing, and each sample is provided with different barcode, thereby data of various samples obtained in data from one sequencing can be differentiated.

Alignment and statistics: Torrent_Server_4.0_VM software is used on raw data generated by PGM sequencer to remove linker sequence and Tmap software is used for alignment to human hg19 reference genome, and haplotype SNP covering multiple and genotype are analyzed finally.

The invention is useful for chromosomal Robertsonian translocation in preimplantation genetic diagnosis on suitable population, and is beneficial for providing genetic counseling and clinical decision basis. The invention is particularly suitable for detecting the condition that one of the parents is chromosomal Robertsonian translocation carrier and the other is normal karyotype, where normal embryo cannot be differentiated from chromosomal Robertsonian translocation carrying embryo according to conventional methods.

The following examples will be provided for detailed description which should not be construed as limitation thereto. The following examples are only parts of the invention, and based on the examples disclosed herein, a person skilled in the art can obtain all other examples which belong to the protection of the invention. Likewise, the accompanying figures are only those of some examples of the invention and based on the figures disclosed herein, a person skilled in the art can obtain all other figures which belong to the protection of the invention.

In case there is no specific description of the experimental conditions in the following examples, they were performed under conventional conditions or those recommended by the manufacture.

EXAMPLE 1 Detection of Embryo Chromosomal Robertsonian Translocation

I. Materials

Sample sources: 8 embryo blastula stage trophoblast cell whole genomic amplified products and parental peripheral blood cell DNA samples, the father carries chromosomal Robertsonian translocation in Chromosome 13 and Chromosome 14 while the mother has normal chromosome karyotype. Analysis results of embryo chromosome copy number are shown in Table 6.

TABLE 6 Parent and embryo chromosome karyotype information No. karyotype Father LH 45, XY, der(13, 14) Mother YT 46, XX Embryo YT_1 46, XY YT_2 46, XX YT_3 47, XY, +14 YT_4 46, XX YT_5 46, XX YT_6 45, XO YT_7 46, XX YT_8 46, XY

II. Procedures

1. Library Constructing and Sequencing

The library was constructed according to Ion AmpliSeg™ Library Kits 2.0 standard Library construction procedures. Multiplex PCR reactions were performed with the primers in Table 1 and Table 2 and then DNA molecules of the amplified products were added with linkers at both ends for sequencing. Under certain conditions, the nucleic acid molecules were allowed to grow in clusters and sequencing were performed on Ion Torrent PGM to give DNA fragments sequences in target location with fragment lengths of 125 bp˜275 bp.

In this example, the sequencing were performed on the DNA samples obtained from the 8 embryo cells and full blood DNA samples obtained from both of the parents according to official sequencing instruction by Ion Torrent.

2. Data Analysis

Torrent_Server_4.0_VM software was used on raw data generated by PGM sequencing to remove linker sequence and Tmap software is used for alignment to human hg19 reference genome. The sequences with low quality were removed and sequencing depth N of target sites were counted and the sites with N<100 were removed. All sequence directions and ratios of each of the bases at valid sites were analyzed. The base type that forward and reverse sequence number >20% and base ratios >10% was designated as one genotype of the site (X, X={A, T, C, G}).

Individual haplotype was speculated with genotypes of the parents and the embryo; while the valid site was the site providing valid SNP information for constructing pedigree haplotype and was generally the site in which one of the parents and the embryo is heterozygous. For the genotypes of the parents, ({X1X1}, {X2X2}), ({X1X1}, {X1X1}) was invalid site, ({X1X1}, {X1X2}) was valid site and ({X1X2}, {X1X2}) was verifiable site. According to valid site and verifiable site, haplotypes of parent and each of embryos were deduced. Male parent (LH), haplotypes of Chromosome 13 and Chromosome 14 were Aa and Bb, respectively and female parent (YT), haplotypes of Chromosome 13 and Chromosome 14 were Cc and Dd, respectively.

III. Results Analysis

Pedigree haplotype was built according to 115 SNP sites in target location (57 and 58 for Chromosome 13 and Chromosome 14, respectively) (results were shown in FIG. 3 and FIG. 4) and pedigree haplotype analysis was shown in Table 7.

TABLE 7 Pedigree haplotype list YT LH YT_1 YT_2 YT_3 YT_4 YT_5 YT_6 YT_7 YT_8 chr13 Cc Aa ac AC AC aC aC Ac AC Ac chr14 Dd Bb bd BD BbD bd bd BD BD Bd

According to the information that LH was translocation carrier and No. 3 embryo had Chromosome 14 duplication, it can be deduced that the Chromosome 13 haplotype from LH (A type) carried by No. 3 embryo was translocation chromosome haplotype. Further, according to other embryo carrying A type or a type, it can be deduced that B type in Chromosome 14 was translocation chromosome haplotype. Accordingly, it can be determined that No. 1, 4, 5 embryos carrying Chromosome 13 a type and Chromosome 14 b type were normal embryos; No. 2, 6, 7, 8 embryos carrying Chromosome 13 A type and Chromosome 14 B type were translocation carrying embryos.

EXAMPLE 2 Detection of Embryo Chromosomal Robertsonian Translocation

I. Materials

Sample sources: 6 embryo blastula stage trophoblast cell whole genomic amplified products and parental peripheral blood cell DNA samples, the father carries chromosomal Robertsonian translocation in Chromosome 13 and Chromosome 14 (JXD) while the mother has normal chromosome karyotype (HCC). Analysis results of embryo chromosome copy number are shown in Table 8.

TABLE 8 Parent and embryo chromosome karyotype information No. karyotype Father JXD 45, XY, der(13; 14) Mother HCC 46, XX Embryo HCC-1 45, XY, −22 HCC-2 45, XY, −13 HCC-3 45, XX, −15 HCC-4 46, XX HCC-5 Not detected HCC-6 46, XY

II. Procedures

1. Library Constructing and Sequencing

The library was constructed according to Ion AmpliSeq™ Library Kits 2.0 standard Library construction procedures. Multiplex PCR reactions were performed with the primers in Table 1 and Table 2 and then DNA molecules of the amplified products were added with linkers at both ends for sequencing. Under certain conditions, the nucleic acid molecules were allowed to grow in clusters and sequencing were performed on Ion Torrent PGM to give DNA fragments sequences in target location with fragment lengths of 125 bp˜275 bp.

In this example, the sequencing were performed on the DNA samples obtained from the 6 embryo cells and full blood DNA obtained from both of the parents according to official sequencing instruction by Ion Torrent.

2. Data Analysis

Torrent_Server_4.0_VM software was used on raw data generated by PGM sequencing to remove linker sequence and Tmap software is used for alignment to human hg19 reference genome. The sequences with low quality were removed and sequencing depth N of target sites were counted and the sites with N<100 were removed. All sequence directions and ratios of each of the bases at valid sites were analyzed. The base type that forward and reverse sequence number >20% and base ratios >10% was designated as one genotype of the site (X, X={A, T, C, G}).

Individual haplotype was speculated with genotypes of the parents and the embryo; while the valid site was the site that providing valid SNP information for constructing pedigree haplotype and was generally the site in which one of the parents and the embryo is heterozygous. For the genotypes of the parents, ({X1X1}, {X2X2}), ({X1X1}, {X1X1}) was invalid site, ({X1X1}, {X1X2}) was valid site and ({X1X2}, {X1X2}) was verifiable site. According to valid site and verifiable site, haplotypes of parent and each of embryos were deduced.

Female parent (HCC), haplotypes of Chromosome 13 and Chromosome 14 were Cc and Dd, respectively and male parent (JXD), haplotypes of Chromosome 13 and Chromosome 14 were Aa and Bb, respectively.

III. Results Analysis

Pedigree haplotype was built according to 115 SNP sites in target location (57 and 58 for Chromosome 13 and Chromosome 14, respectively) (results were shown in FIG. 1) and pedigree haplotype analysis was shown in Table 9.

TABLE 9 Pedigree haplotype list HCC JXD HCC-1 HCC-2 HCC-3 HCC-4 HCC-5 HCC-6 chr13 Cc Aa AC C aC AC — ac chr14 Dd Bb BD bD bD BD — bD

According to the information that JXD is translocation carrier and No. 2 embryo had Chromosome 13 deletion, it can be deduced that the Chromosome 14 haplotype from JXD (b type) carried by No. 2 embryo was normal chromosome haplotype, i.e. Chromosome 14 B type was translocation chromosome haplotype. Further, according to other embryo carrying B type or b type, it can be deduced that A type in Chromosome 13 was translocation chromosome haplotype. Accordingly, it can be determined that No. 1, 4 embryos carrying Chromosome 14 B type and Chromosome 13 A type were translocation carrier embryos; No. 3, 6 embryos carrying Chromosome 14 b type and Chromosome 13 a type were normal embryos. No result was detected for No. 5 embryo chromosome copy number analysis and there was no valid sequence for this detection.

INDUSTRIAL APPLICABILITY OF THE INVENTION

The invention is useful for embryo chromosomal translocation analysis on suitable population, and is beneficial for providing genetic counseling and clinical decision basis. A preimplantation genetic diagnosis can effectively prevent children patients from birth.

The suitable population according to the invention may be chromosomal Robertsonian translocation carriers determined by routine chromosome karyotype analysis.

The invention is not limited to the best embodiments as listed above. Anybody can achieve other products in various forms under the inspiration of the invention. The technical solution, no matter changes to the shape or structure, which is similar or identical to the invention, falls within the protection scope of the invention. 

The invention claimed is:
 1. A method for genotyping SNPs involved in chromosome Robertsonian translocation (“CRT”), the method comprising: (a) obtaining a CRT-carrier parent DNA sample, a non-CRT-carrier parent DNA sample, and a progeny DNA sample from a progeny of the CRT-carrier and the non-CRT-carrier parents; (b) performing PCR amplification on the obtained samples from both of the parents and the progeny to produce amplicons, wherein the PCR amplification uses each of the following primer pairs: (i) SEQ ID NO: 1 and SEQ ID NO: 2; (ii) SEQ ID NO: 3 and SEQ ID NO: 4; (iii) SEQ ID NO: 115 and SEQ ID No: 116; (iv) SEQ ID NO: 117 and SEQ ID NO: 118; (v) SEQ ID NO: 231 and SEQ ID NO: 232; (vi) SEQ ID NO: 233 and SEQ ID NO: 234; (vii) SEQ ID NO: 379 and SEQ ID NO: 380; (viii) SEQ ID NO: 381 and SEQ ID NO: 382; (ix) SEQ ID NO: 529 and SEQ ID NO: 530; and (x) SEQ ID NO: 531 and SEQ ID NO: 532; and (c) using the amplicons to genotype the SNPs, wherein the SNPs are located at the following genomic positions in the human reference genome hg19: chr13:19602195, chr13: 19625370, chr14: 19472367, chr14:20326905, chr15:20022190, chr15:20049922, chr21: 14640496, chr21: 14643423, chr22:16415691, and chr22:16554800.
 2. The method according to claim 1, wherein the CRT-carrier parent or the non-CRT-carrier parent DNA sample is extracted from a peripheral blood sample, while the progeny DNA sample is extracted an embryo, a fetus, or an abortion tissue.
 3. The method according to claim 1, wherein the progeny is Robertsonian translocation chromosome trisomic or monosomic. 